Methods of diagnosing and treating small intestinal bacterial overgrowth (SIBO) and SIBO-related conditions

ABSTRACT

Disclosed is a method of treating small intestinal bacterial overgrowth (SIBO) or a SIBO-caused condition in a human subject. SIBO-caused conditions include irritable bowel syndrome, fibromyalgia, chronic pelvic pain syndrome, chronic fatigue syndrome, depression, impaired mentation, impaired memory, halitosis, tinnitus, sugar craving, autism, attention deficit/hyperactivity disorder, drug sensitivity, an autoimmune disease, and Crohn&#39;s disease. Also disclosed are a method of screening for the abnormally likely presence of SIBO in a human subject and a method of detecting SIBO in a human subject. A method of determining the relative severity of SIBO or a SIBO-caused condition in a human subject, in whom small intestinal bacterial overgrowth (SIBO) has been detected, is also disclosed.

This application is a continuation of U.S. patent application Ser. No.11/348,995, filed Feb. 7, 2006 and issued as U.S. Pat. No. 7,736,622,which is a divisional of U.S. patent application Ser. No. 09/837,797,filed Apr. 17, 2001 and issued as U.S. Pat. No. 7,048,906 on May 23,2006, which is a continuation-in-part of U.S. patent application Ser.No. 09/374,142, filed on Aug. 11, 1999 and issued as U.S. Pat. No.6,861,053 on Mar. 1, 2005, a continuation-in-part of U.S. patentapplication Ser. No. 09/374,143, filed on Aug. 11, 1999 and issued asU.S. Pat. No. 6,562,629 on May 13, 2003, and a continuation-in-part ofU.S. patent application Ser. No. 09/546,119, filed on Apr. 10, 2000 andissued as U.S. Pat. No. 6,558,708 on May 6, 2003, which is acontinuation-in-part of U.S. patent application Ser. No. 09/420,046,filed Oct. 18, 1999, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/359,583, filed on Jul. 22, 1999, abandoned,which was a continuation of U.S. patent application Ser. No. 08/832,307,filed on Apr. 3, 1997 and issued as U.S. Pat. No. 5,977,175 on Nov. 2,1999, which was a continuation of U.S. patent application Ser. No.08/442,843, filed on May 17, 1995, abandoned.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant NIH DK46459.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced withinparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

1. The Field of the Invention

This invention relate to the medical arts. It relates to a method ofdiagnosing and treating small intestinal bacterial overgrowth (SIBO),and conditions caused by SIBO.

2. Discussion of the Related Art

Small intestinal bacterial overgrowth (SIBO), also known as small bowelbacterial overgrowth (SBBO), is an abnormal condition in which aerobicand anaerobic enteric bacteria from the colon proliferate in the smallintestine, which is normally relatively free of bacterial contamination.SIBO is defined as greater than 10⁶ CFU/mL small intestinal effluent (R.M. Donaldson, Jr., Normal bacterial populations of the intestine andtheir relation to intestinal function, N. Engl. J. Med. 270:938-45[1964]). Typically, the symptoms include abdominal pain, bloating, gasand alteration in bowel habits, such as constipation and diarrhea.

Irritable bowel syndrome, Crohn's disease, chronic fatigue syndrome,chronic pelvic pain syndrome, fibromyalgia, depression, attentiondeficit/hyperactivity disorder, autism, and autoimmune diseases, e.g.,multiple sclerosis and systemic lupus erythematosus, are all clinicalconditions of unclear etiology. No association has been Made heretoforebetween any of the afore-going diagnostic categories and SIBO.

Irritable bowel syndrome (IBS) is the Most common of allgastrointestinal disorders, affecting 11-14% of adults and accountingfor more than 50% of all patients with digestive complaints. (G.Triadafilopoulos et al., Bowel dysfunction in fibromyalgia, DigestiveDis. Sci. 36(I):59-64 [1991]; W. G. Thompson, Irritable Bowel syndrome:pathogenesis and management, Lancet 341:1569-72 [1993]). It is thoughtthat only a minority of people with IBS actually seek medical treatment.Patients with IBS present with disparate symptoms, for example,abdominal pain predominantly related to defecation, alternating diarrheaand constipation, abdominal distention, gas, and excessive mucus in thestool.

A number of possible causes for IBS have been proposed, but none hasbeen fully accepted. (W. G. Thompson [1993]). These hypotheses includeda fiber-poor Western diet, intestinal motility malfunction, abnormalpain perception, abnormal psychology or behavior, or psychophysiologicalresponse to stress.

A high fiber diet increases stool bulk and shortens gut transit time.However the presence of IBS in non-Western countries, such as China andIndia, and the failure of dietary fiber supplements to treat IBS indouble-blind clinical trials are inconsistent with the “fiberhypothesis” for the causation of IBS. (W. Bi-zhen and P. Qi-Ying,Functional bowel disorders in apparently healthy Chinese people; Chin.J. Epidemiol. 9:345-49 [1988]; K. W. Heaton, Role of dietary fibre inirritable bowel syndrome. In: R. W. Read [ed.], Irritable bowelsyndrome, Grune and Stratton, London, pp. 203-22 [1985]; W. G. Thompsonet al., Functional bowel disorders and functional abdominal pain,Gastroenterol. Int. 5:75-92 [1992]).

Those experiencing chronic IBS pain are often depressed and anxious.Treatment with tricyclic antidepressants has been used to raise the painthreshold of some IBS patients (W. G. Thompson [1993]). Abreu et al. andRabinovich et al. taught the use of corticotropin-releasing factorantagonists to relieve stress-related symptoms, including depression andanxiety, in IBS, anorexia nervosa, and other disorders. (M. E. Abreu,Corticotropin-releasing factor antagonism compounds, U.S. Pat. No.5,063,245; A. K. Rabinovich et al., Benzoperimidine-carboxylic acids andderivatives thereof; U.S. Pat. No. 5,861,398). Becker et al. taught theuse of serotonin antagonists to treat depression and anxiety associatedwith IBS and other conditions. (D. P Becker et al., Meso-azacyclicaromatic acid amides and esters as serotonergic agents, U.S. Pat. No.5,612,366).

Those with IBS symptoms have not been shown to have differentpsychological or psychosocial make-up from the normal population. (W. E.Whitehead et, al., Symptoms of psychologic distress associated withirritable bowel syndrome: comparison of community and medical clinicsamples, Gastroenterol. 95:709-14 [1988]). But many IBS patients appearto perceive normal intestinal activity as painful. For example, IBSpatients experience pain at tower volumes of rectal distention thannormal or have a lower than normal threshold for perceiving migratingmotor complex phase III activity. (W. E. Whitehead et al., Tolerance forrectosigmoid distension in irritable bowel syndrome, Gastroenterol:98:1187-92 [1990]; J. E. Kellow et al., Enhanced perception ofphysiological intestinal motility in the irritable bowel syndrome,Gastroenterol. 101(6): 1621-27 [1991]).

Bowel motility in IBS patients differs from normal controls in responseto various stimuli such as drugs, hormones, food, and emotional stress.(D. G. Wangel and D. J. Deller, Intestinal motility in man, III:mechanisms of constipation and diarrhea with particular reference to theirritable bowel, Gastroenterol. 48:69-84 [1965]; R. F. Harvey and A. E.Read, Effect of cholecystokinin on colon motility on and symptoms inpatients with irritable bowel syndrome, Lancet i:1-3 [1973]; R. M.Valori et al., Effects of differents types of stress and “prokineticdrugs” on the control of the fasting motor complex in humans,Gastroenterol 90:1890-900 [1986]).

Evans et al. and Gorard and Farthing recognized that irritable bowelsyndrome is frequently associated with disordered gastro-intestinalmotility. (P. R Evans et al., Gastroparesis and small bowel dysmotilityin irritable bowel syndrome, Dig. Dis. Sci. 42(10):2087-93 [1997]; D A.Gorard and M. J. Farthing, Intestinal motor function in irritable bowelsyndrome, Dig. Dis. 12(2):72-84 [1994]). Treatment directed to boweldysmotility in IBS includes the use of serotonin antagonists (D. PBecker et al., Meso-azacyclic aromatic acid amides and esters asserotonergic agents, U.S. Pat. No. 5,612,366; M. Ohta et al., Method oftreatment of intestinal diseases, U.S. Pat. No. 5,547,961) andcholecystokinin antagonists (Y. Sato et al., Benzodiazepine derivatives,U.S. Pat. No. 4,970,207; H. Kitajima et al., Thienylazole compound andthienotriozolodiazepine compound, U.S. Pat. No. 5,760,032). But colonicmotility index, altered myoelectrical activity in the colon, and smallintestinal dysmotility have not proven to be reliable diagnotic tools;because they are not IBS-specific. (W. G. Thompson [1993]).

Because there has been no known underlying cause for IBS, treatment ofIBS has been primarily directed to symptoms of pain, constipation ordiarrhea symptoms.

For example, administration of the polypeptide hormone relaxin, used torelax the involuntary muscles of the intestines, is a treatment taughtto relieve the pain, associated with IBS. (S. K. Yue, Method of treatingmyofascial pain syndrome with relaxin, U.S. Pat. No. 5,863,552).

Borody et al. taught the use of a picosulfate-containing laxativepreparation to treat constipation in IBS, small intestinal bacterialovergrowth, and acute or chronic bacterial bowel infections. (T. J.Borody et al., Picosulfate-containing preparation for colonicevacuation, U.S. Pat. No. 5,858,403). Barody also taught the use of ananti-inflammatory agent to treat IBS. (T. J. Barody, Treatment ofnon-inflammatory and non-infectious bowel disorders, U.S. Pat. No.5,519,014). In addition, constipation in IBS has been treated withamidinourea compounds. (J. Yelnosky et al., Amidinoureas for treatingirritable bowel syndrome, U.S. Pat. Nos. 4,701,457 and 4,611,011).

Kuhla et al. taught the use of triazinone compounds to relieve IBSsymptoms of constipation, diarrhea, and abdominal pain (D. E. Kuhla etal., Triazinones for treating irritable bowel syndrome U.S. Pat. No.4,562,188): And Kitazawa et al. taught the use of napthy- andphenyl-sulfonylalkanoic acid compounds to treat IBS symptom. (M.Kitazawa et al., Naphthysulfonylalkanoic acid compounds andpharmaceutical compositions thereof, U.S. Pat. No. 5,177,069; M.Kitazawa et al., Phenylsulfonylalkanoic acid compounds andpharmaceutical compositions thereof, U.S. Pat. No. 5,145,869). Daytaught an IBS treatment involving the administration of an anion-bindingpolymer and a hydrophilic-polymer, (C. E. Day, Method for treatment ofirritable bowel syndrome, U.S. Pat. No. 5,380,522). And Borody et al.taught the use of salicylic acid derivatives to treat IBS. (T. J. Borodyet. al., Treatment of non-inflammatory and bowel disorders, U.S. Pat.No. 5,519,014).

A probiotic approach to the treatment of IBS has also been tried. Forexample, Allen et al. described the use of a strain of Enterococcusfaecium to alleviate symptoms. (W. D. Allen et al., Probiotic containingEnterococcus faecium Strain NCIMB 40371, U.S. Pat. No. 5,728,380 andProbiotic, U.S. Pat. No. 5,589,168). Borody taught a method of treatingirritable bowel syndrome by at least partial removal of the existingintestinal microflora by lavage and replacement with a new bacterialcommunity introduced by fecal inoculum from a disease-screened humandonor or by a composition comprising Bacteroides and Escherichia colispecies. (T. J. Borody, Treatment of gastro-intestinal disorders with afetal composition or a composition of bacteroides and E. coli, U.S. Pat.No. 5,443,826).

Fibromyalgia (FM) is a syndrome of intense generalized pain andwidespread local tenderness, usually associated with morning stiffness,fatigue, and sleep disturbances. (F. Wolfe, Fibromyalgia: the clinicalsyndrome, Rheum. Dis. Clin. N. Amer. 15(1)1-17 [1989]). Fibromyalgia isoften associated with IBS (34-50% of FM: caset) or othergastrointestinal symptoms, Raynaud's phenomenon, headache, subjectiveswelling, paresthesias, psychological abnormality or functionaldisability; sometimes with overlapping symptoms of coexisting arthritis,lower back and cervical disorders, and tendonitis. Fibromyalgia affects1-5% of the population and is more prevalent among women than men. (G.Triadafilopoulos et al. [1991]).

As in IBS, a diagnosis of FM correlates with a decreased pain thresholdamong FM patients compared to non-patients. (F. Wolfe et al., Aspects ofFibromyalgia in the General Population: Sex, Pain Threshold andFibromyalgia Symptoms, J. Rheumatol. 22:151-56 [1995]). But otherconventional laboratory evaluations of FM patients are uniformly normal.(G. Triadafilopoulos et al. [1991]). The symptoms of FM patients aretypically treated with anti-inflammatory agents and low dose tricyclicantidepressants. Administration of relaxin for involuntary muscledysfunction is also a treatment taught to relieve the pain associatedwith fibromyalgia. (S. K. Yue, Method of treating myofascial painsyndrome with relaxin, U.S. Pat. No. 5,863,552). However, there has beenno know cause of FM to which diagnosis and/or treatment could bedirected.

Chronic fatigue syndrome (CFS) affects more than a half millionAmericans. (P. H. Levine; What we know about chronic fatigue syndromeand its relevance to the practicing physician, Am. J. Med.105(3A):100S-03S [1998]). Chronic fatigue syndrome is characterized by asudden onset of persistent, debilitating fatigue and energy loss thatlasts at least six months and cannot be attributed to other medical orpsychiatric conditions, symptoms include headache, cognitive andbehavioral impairment, sore throat, pain in lymph nodes and joints, andlow grade fever. (M. Terman et al., Chronic Fatigue Syndrome andSeasonal; Affective Disorder: Comorbidity, Diagnostic Overlap, andImplications for Treatment, Am. J. Med. 105(3A):115S-24S [1998]).Depression and related symptoms are also common, including sleepdisorders, anxiety, and worsening of premenstrual symptoms or othergynecological complications. (A. L. Komaroff and D. Buchwald, Symptomsand signs of chronic fatigue syndrome, Rev. Infect. Dis. 13:S8-S11[1991]; B. L. Harlow et al., Reproductive correlates of chronic fatiguesyndrome, Am. J. Med. 105(3M:94S-99S [1998]). Other physiologicabnormalities are also associated with CFS in many patients, includingneurally-mediated, hypotension, hypocortisolism, and immunologicdysregulation (P. H. Levine [1998]). A subgroup of CFS patients complainof exacerbated mood state, diminished ability to work and difficultyawakening during winter months, reminiscent of seasonal affectivedisorder. (M. Terman et al. [1998]).

The etiology of CFS has been unknown, and the hetetogeneity of CFSsymptoms has precluded the use of any particular diagnostic laboratorytest. (P. H. Levine [1998]). Symptomatic parallels have been suggestedbetween CFS and a number of other disease conditions, resulting fromviral infection, toxic exposure, orthostatic hypotension, and stress,but node of these has been shown to have a causal role in CFS. (E.g., I.R. Bell, et al., Illness from low levels of environmental chemicals:relevance to chronic fatigue syndrome and fibromyalgia, Am. J. Med.105(3A)74S-82S [1998]; R. L. Bruno et al., Parallels between post-poliofatigue and chronic fatigue syndrome: a common pathophsiology?, Am. J.Med. 105(3A):66S-73S [1998]; R. Glaser and J. K. Kiecolt-Glaser;Stress-associated immune modulation: relevance to viral infections andchronic fatigue syndrome, Am. J. Med. 105(3A) 35S-42S. [1998]; P. C.Rowe and H. Calkins, Neurally mediated hypotension and chronic fatiguesyndrome, Am. J. Med. 105(3A):15S-21S [1998]; L. A. Jason et al.,Estimating the prevalence of chronic fatigue syndrome among nurses, Am.J. Med. 105(3A):91S-93S [1998]). One study reported that there was nosupport for an etiological role in CFS of Yersinia enterocoliticainfection. (C. M. Swanink et al., Yersinia entercolitica and the chronicfatigue syndrome, J. Infect. 36(3):269-72 [1998]). Accordingly, therehas been no known cause to which diagnosis and/or treatment of CSF couldbe directed.

Consequently, the diagnosis and treatment of CFS have continued to bedirected to symptoms, rather than to an underlying treatable cause. Forexample, the use of relaxin has been described for relaxing theinvoluntary muscles and thus relieve pain associated with CFS: (S. K.Yue, Method of treating myofascial pain syndrome with relaxin, U.S. Pat.No. 5,863,552).

Attention deficit/hyperactivity disorder (ADHD) is a heterogeneousbehaviorial disorder of unknown etiology that always appears first inchildhood, affecting 3-20% of elementary school-age children, andcontinues to affect up to 3% of adults. (Reviewed in L. L. Greenhill,Diagnosing attention deficit/hyperactivity disorder in children, J. ClinPsychiatry 59 Suppl 7:31-41 [1998]). Those affected with ADHD symptomstypically exhibit inattentiveness and distractability (AD type),hyperactive and impulsive behavior (HI type), or a combination of these,to a degree that impairs normal functioning and is often sociallydisruptive. (M. L. Wolraich et al., Examination of DSM-IV criteria forattention deficit/hyperactivity disorder in a county-wide sample, J.Dev. Behav. Pediatr. 19(3):162-68 [1998]; J. J. Hudziak et al., Latentclass and factor analysis of DMS-IV ADHD: a twin study of femaleadolescents, J. Am. Acad. Child Adolesc. Psychiatry37(8):848-57 [1998]).Often prescribed are central nervous system stimulants, tricyclicantidepressants, antihypertensives, analgesics, or antimanic drugs, butthere has been no known cause of ADHD to which diagnosis and/ortreatment could be directed. (S. C. Schneider and G. Tan, Attentiondeficit/hyperactivity disorder. In pursuit of diagnostic accuracy;Postgrad. Med. 101(4)131-2, 235-40 [1997]; W. J. Barbaresi, Primary-careapproach 16 the diagnosis and management of attentiondeficit/hyperactivity disorder, Mayo Clin. Proc. 7.1(5):46371 [1996]).

There has also been no known cause for autoimmune diseases, includingmultiple sclerosis and systemic lupus erythematosus. Multiple sclerosis(MS) is a neurologic disease that primarily strikes teens and youngadults under 35 years. Affecting 350,000 Americans, MS is the mostfrequent cause of neurologic disability except for traumatic injuries;MS affects twice as many females compared to males. (S. L. Hauser,Multiple Sclerosis and other demyelinating diseases In: Harrison'sPrinciples of Internal Medicine, 13th ed., K. J. Isselbacher et al.(eds.), McGraw-Hill, pp. 2287-95 [1994]). The disease is characterizedby chronic inflammation, scarring, and selective destruction of themyelin sheath around neural axons of the central nervous system, and isthought to be caused by autoimmune responses. A treatment for MS taughtby Weiner et al. is related to oral administration of autoantigens tothe patient to suppress the autoimmune response, by eliciting suppressorT-cells specific for myelin basic protein (MBP). There are no specificdiagnostic tests for MS; diagnosis is based on clinical recognition ofdestructive patterns of central nervous system injury that are producedby the disease. (S. L. Hauser [1994]) Nerve damage may be mediated bycyokines, especially TNF-α, which has been found be selectively toxic tomyelin and to oligodendrocytes in vitro. Elevated levels of TNF-α andIL-2 were measured in MS patients. (J. L. Trotter et al., Serum cytokinelevels in chronic progressive multiple sclerosis: interleukin-2 levelsparallel tumor necrosis factor-alpha levels, J. Neuroimmunol.33(1):29-36 [1991]; H. L. Weiner et al., Treatment of multiple sclerosisby oral administration of autoantigens, U.S. Pat. No. 5,869,054).Another treatment for MS involves the administration of a vitamin Dcompound. (H. F. DeLuca et al., Multiple sclerosis treatment, U.S. Pat.No. 5,716,946). However, there has been no known cause of MS to whichdiagnosis and/or treatment could be directed.

Systemic lupus erythematosus (SLE) is an autoimmune rheumatic diseasecharacterized by deposition in tissues of autoantibodies and immunecomplexes leading to tissue injury (B. L. Kotzin, Systemic lupuserythematosus, Cell 85:303-06 [1996]). In contrast to autoimmunediseases such as MS and type 1 diabetes mellitus, SLE potentiallyinvolves multiple organ systems directly, and its clinicalmanifestations ate diverse and variable. (Reviewed by B. L. Kotzin andJ. R. O'Dell, Systemic lupus erythematosus, In: Samler's ImmunologicDiseases, 5th ed., M. M. Frank et al., eds, Little Brown & Co., Boston,pp. 667-97 [1995]). For example, some patients may demonstrate primarilyskin rash and joint pain, show spontaneous remissions, and requirelittle medication. At the other end of the spectrum are patients whodemonstrate severe and progressive kidney involvement that requirestherapy with high doses of steroids and cytotoxic drugs such ascyclophosphamide (B. L. Kotzin [1996]).

The serological hallmark of SLE, and the primary diagnostic testavailable, is elevated serum levels of IgG antibodies to constituents ofthe cell nucleus, such as double-stranded DNA (dsDNA), single-strandedDNA (ss-DNA), and chrormatin. Among these autoantibodies, IgG anti-dsDNAantibodies play a major role in the development of lupusglomerulonephritis (GN). (B. H. Hahn and B. Tsao, Antibodies to DNA, In:Dubois' Lupus Erythematosus, 4th ed., D. J. Wallace and R. Hahn, eds.,Lea and Febiger, Philadelphia, pp. 195-201 [1993]; OhniShi et al.,Comparison of pathogenic and nonpathogenic murine antibodies to DNA:Antigen binding and structural characteristics, Int. Immunol. 6:817-30[1994]). Glomerulonephritis is a serious condition in which thecapillary waifs of the kidney's blood purifying glomeruli becomethickened by accretions on the epithelial side of glomerular basementmembranes. The disease is often chronic and progressive and may lead toeventual renal failure.

The mechanisms by which autoantibodies are induced in these autoimmunediseases remains: unclear. As there has been no known cause of SLE, towhich diagnosis and/or treatment could be directed, treatment has beendirected to suppressing immune responses; for example with macrolideantibiotics, rather than to an underlying cause. (E.g., Hitoshi et al.,Immunosuppressive agent, U.S. Pat. No. 4,843,092).

Another disorder for, which immunosuppression has been tried is Crohn'sdisease. Crohn's disease symptoms include intestinal inflammation andthe development of intestinal stenosis and fistulas; neuropathy, oftenaccompanies these symptoms. Anti-inflammatory drugs, such as5-aminosalicylates (e.g., mesalamine) or corticosteroids, are typicallyprescribed, but are not always effective. (Reviewed in V. A. Botoman etal., Management of Inflammatory Bowel Disease, Am. Fam. Physician57(1):57-68 [1998]). Immunosuppression with cyclosporine is sometimesbeneficial for patients resistant to or intolerant of corticosteroids:(J. Brynskov et al., A placebo-controller, double-blind, randomizedtrial of cyclosprorine therapy in active chronic Crohn's disease, N.Engl. J. Med. 321(13):845-50 [1989]).

Nevertheless, surgical correction is eventually required in 90% ofpatients, 50% undergo colonic resection. (K. Leipet et al., Adjuvantpost-operative therapy, Baillieres Clin. Gastroenterol. 12(I):179-99[1998]; F. Makowiec et al., Long-term follow-up after resectionalsurgery in patients with Crohn's disease involving the colon, Z.Gastroenterol. 36(8):619-24 [1998]). The recurrence rate after surgeryis high, with 50% requiring further surgery within 5 years. (K. Leiperet al., [1998]; M. Besnard et al., Postoperative outcome of Crohn'sdisease in 30 children, Gut 43(5):634-38 [1998]).

One hypothesis for the etiology of Crohn's disease is that a failure ofthe intestinal mucosal barrier, possibly resulting from geneticsusceptibilities and environmental factors (e.g., smoking), exposes theimmune system to antigens from the intestinal lumen including bacterialand food antigens (e.g., Söderholm et al., Epithelial permeability toproteins in the non-inflamed ileum of Crohn's disease?, Gastroenterol.117:65-72 [1999]; D. Hollander et al., Increased intestinal permeabilityin patients with Crohn's disease and their relatives. A possibleetiologic factor, Ann. Intern. Med. 105:883-85 [1986]; D. Hollander, Theintestinal permeability barrier. A hypothesis to its involvement inCrohn's disease, Scand. J. Gastroenterol. 27:721-26 [1992]). Anotherhypothesis is that persistent intestinal infection by pathogens such asMycobacterium paratuberculosis, Listeria Monocytogenes, abnormalEscherichia coli, or paramyxovirus, stimulates the immune response; oralternatively, symptoms result from a dysregulated immune response toubiquitous antigens, such as normal intestinal microflora and themetabolites and toxins they produce. (R. B. Sartor, Pathogenesis andImmune Mechanisms of Chronic Inflammatory Bowel Diseases, Am. J.Gastroenterol. 92(12):5S-11S [1997]). The presence of IgA and IgGanti-Sacccharomyces cerevisiae antibodies (ASCA) in the serum was foundto be highly diagnostic of pediatric Crohn's disease (F. M. Ruemmele etal., Diagnostic accuracy of serological assays in pediatric inflammatorybowel disease, Gastroenterol. 115(4):822.29 [1998]; E. J. Hoffenberg etal., Serologic testing for inflammatory, bowel disease, J. Pediatr.134(4):447-52 [1999]).

In Crohn's disease, a dysregulated immune response is skewed towardcell-mediated immunopathology. (S. I. Murch, Local and systemic effectsof macrophage cytokines in intestinal inflammation, Nutrition 14:780-83[1998]): But immunosuppressive drugs, such as cyclosporine, tacrolimus,and mesalamine have been used to treat corticosteroid-resistant cases ofCrohn's disease with mixed success. (J. Brynskov et al. [1989]; K.Fellerman et al., Steroid-unresponsive acute attacks of inflammatorybowel disease: immunomodulation by tacrolimus [FK506], Am. J.Gastroenterol. 93(10):1860-66 [1998]). An abnormal increase in colonicpermeability is also seen in patients with Crohn's disease. (Vermeire S.et al., Anti-Saccharomyces cerevisiae antibodies (ASCA), phenotypes ofIBD, and intestinal permeability: a study in IBD families, Inflamm towelDis. 7(1)845 [2001]).

Recent efforts to develop diagnostic and treatment tools against Crohn'sdisease have focused on the central role of cytokines. (S. Schreiber,Experimental immunomodulatory therapy of inflammatory bowel disease,Neth. J. Med. 53(6)S24-31 [1998]; R. A. van Hogezand and H. W.Verspaget, The future role of anti-tumor necrosis factor-alpha productsin the treatment of Crohn's disease, Drugs 56(3):299-305 [1998]).Cytokines are small secreted proteins or factors (5 to 20 kD) that havespecific effects on cell-to-cell interactions, intercellularcommunication, or the behavior of other cells. Cytokines are produced bylymphocytes, especially T_(H)1 and T_(H)2 lymphocytes, monocytes,intestinal macrophages, granulocytes, epithelial cells, and fibroblasts.(Reviewed in, G. Rogler and T. Andus, Cytokines in inflammatory boweldisease, World J. Surg. 22(4):382-89. [1998]; H. F. Galley and N. R.Webster, The immuno-inflammatory cascade, Br. J. Anaesth. 77:11-16[1996]). Some cytokines are pro-inflammatory (e.g., tumor necrosisfactor [TNF]-α, interleukin [IL]-1(α and β), IL-6, IL4, IL-12, orleukemia inhibitory factor [LIF]), others are anti-inflammatory (e.g.,IL-1 receptor antagonist [IL-1ra], IL-4, IL-10, IL-11, and transforminggrowth factor [TGF]-β). However, there may be overlap and functionalredundancy in their effects under certain inflammatory conditions.

In active cases of Crohn's disease, elevated concentrations of TNF-α andIL-6 are secreted into the blood circulation, and TNF-α, IL-1, IL-6; andIL-8 are produced in excess locally by mucosal cells. (Id.; K. Funakoshiet al. Spectrum of cytokine gene expression in intestinal mucosallesions of Crohn disease and ulcerative colitis, Digestion 59(1):73-78[1998]). These cytokines can have far-ranging effects on physiologicalsystems including bone development, hematopoiesis, and liver, thyroid,and neuropsychiatric function. Also, an imbalance of the IL-1β/IL-1raratio, in favor of pro-inflammatory IL-1β, has been observed in patientswith Crohn's disease. (G. Rogler and T. Andus. [1998]; T. Saiki et al.,Detection of pro- and anti-inflammatory cytokines in stools of patientswith inflammatory bowel disease, Scand. J. Gastroenterol. 33(6):616-22[1998]; S. Dionne et al., Colonic explant production of IL-1 and itsreceptor antagonist is imbalanced in inflammatory bowel disease (IBD),Clin. Exp. Imunol. 112(3):435-42 [1998]; But see S. Kuboyama, Increasedcirculating levels of interleukin-1 receptor antagonist in patients withinflammatory bowel disease, Kurume Med. J. 45(1):33-37 [1998]). Onestudy suggested that cytokine profiles in stool samples could be auseful diagnostic tool for Crohn's disease. (T. Saiki et al. [1998]).

Treatments that have been proposed for Crohn's disease include the useof various cytokine antagonists (e.g., IL-1ra), inhibitors (e.g., ofIL-β converting enzyme and antioxidants) and anti-cytokine antibodies.(G. Rogler and T. Andus [1998]; R A. van Hogezand and H. W. Verspaget[1998]; J. M. Reimund et al., Antioxidants inhibit the in vitroproduction of inflammatory cytokines in Crohn's disease and ulcerativecolitis, Eur. J. Clin. Invest. 28(2):145-50 [1998]; N. Lugering et al.,Current concept of the role of monocytes/macrophages in inflammatorybowel disease-balance of pro-inflammatory and immunosuppressivemediators, Ital. J. Gastroenterol. Hepatol 30(3)338-44 [1998]; M. E.McAlindon et al., Expression of interleukin 1 beta and interleukin 1beta converting enzyme by intestinal macrophages in health andinflammatory bowel disease, Gut 42(2):214-19 [1998]). In particular,monoclonal antibodies against TNF-α have been tried with some success inthe treatment of Crohn's disease. (S. R. Targan et al., A short-termstudy of chimeric monoclonal antibody cA2 to tumor necrosis factor alphafor Crohn's disease. Crohn's Disease cA2 Study Group, N. Engl. J. Med.337(15):1029-35 [1997] W. A. Stack et al., Randomised controlled trialof CDP571 antibody to tumour necrosis factor-alpha in Crohn's disease,Lancet 349(9051):521-24 [1997]; H. M. van Dullemen et al., Treatment ofCrohn's disease with anti-tumor necrosis factor chimeric monoclonalantibody, (cA2), Gastroenterol. 109(1):129-35 [1995]).

Another approach to the treatment of Crohn's disease has focused on atleast partially eradicating the bacterial community that may betriggering the inflammatory response and replacing it with anon-pathogenic community. For example, McCann et al (McCann et al.,Method for treatment of idiopathic inflammatory bowel disease, U.S. Pat.No. 5,599,795) disclosed a method for the prevention and treatment ofCrohn's disease in human patients. Their method was directed tosterilizing the intestinal tract with at least one antibiotic and atleast one anti-fungal agent to kill off the existing flora and replacingthem with different, select, well-characterized bacteria taken fromnormal humans. Borody taught a method of treating Crohn's disease by atleast partial removal of the existing intestinal microflora by lavageand replacement with a new bacterial community introduced by fecalinoculum from a disease-screened human donor or by a compositioncomprising Bacteroides and Escherichia coli species. (T. J. Barody,Treatment of gastro-intestinal disorders with a fecal composition or acomposition of bacteroides and E. coli, U.S. Pat. No. 5,443,826).However, there has been no known cause of Crohn's disease to whichdiagnosis and/or treatment could be directed.

Pain is a common symptom associated with irritable bowel syndrome,fibromyalgia, chronic fatigue syndrome, chronic pelvic pain syndrome,depression, ADHD, autoimmune diseases, and Crohn's disease. While theexperience of pain is intertwined with a person's emotions, memory,culture, and psychosocial situation (D. A. Drossman and W. G. Thompson,Irritable bowel syndrome: a graduated multicomponent treatment approach,Ann. Intern. Med. 116:1009-16 [1992]), evidence shows that certaincytokine mediated-immune responses can influence the perception of pain.Cytokines can be released in response to a variety of irritants and canmodulate the perception of pain. For example, exposure of humanbronchial epithelial cells to irritants, including acidic pH, results ina receptor, mediated release of inflammatory cytokines IL-6, IL-8, andTNF-α. (B. Veronesi et al., Particulate Matter initiates inflammatorycytokine release by activation of capsaicin and acid receptors in ahuman bronchial epithelial cell line, Toxicol. Appl. Pharmacol:154:106-15 [1999]). Irritant receptors on cell surfaces, e.g., receptorssensitive to noxious stimuli, such as capsaicin and pH, mediate therelease of cytokines and also mediate the release of neuropeptides fromsensory nerve fibers, which is known to result in a neurogenicinflammatory processes and hyperalgesia (excessive sensitivity to pain).(Id.; R. O. P. de Campos et al., Systemic treatment with Mycobacteriumbovis bacillus calmett-guerin (BCG) potentiates kinin B ₁ receptoragonist-induced nociception and oedema formation in the formalin test inmice, Neuropeptides 32(5): 393-403 [1998]).

The perception of pain, is also influenced by the mediation of kinin B₁and B₂ receptors, which bind peptides called kinins, e.g., thenonapeptide bradykinin or the decapeptide kallidin (lysyl bradykinin).While the precise mechanism of action is unknown, kinins cause therelease of other pro-inflammatory and hyperalgesic mediators such asneuropeptides. Cytokines IL-1(α and β), IL-2, IL-6, and TNF-α arethought to activate kinin B₁ receptor, and thus can contribute toenhanced perception of pain. (R. O. P. de Campos et al. [1998]. Theendotoxin of Escherichia coli significantly activated kinin B₁receptor-mediated neurogenic and inflammatory pain responses in animals.(M M. Campos et al., Expression of B ₁ kinin receptors mediating pawoedema formalin-induced nociception. Modulation by glucocorticoids, Can.J. Physiol. Pharmacol. 73:812-19 [1995]):

It has also been shown that IL-1β, IL-6, and TNF-α, administered to themammalian brain, can modulate pain perception viaprostaglandin-dependent processes. (T. Hori et al., Pain modulatoryactions of cytokines and prostaglandin E ₂ in the Brain, Ann. N.Y. Acad.Sci. 840:269-81 [1.998]). Granulocytes, which accumulate in nearly allforms of inflammation, are non-specific amplifiers and effectors ofspecific immune responses, and they can also modulate the perception ofpain. Neutrophils, a type of granulocyte cell, are known to accumulatein response to IL-1β, and neutrophil accumulation plays a crucialpositive role in the development of nerve growth factor (NGF)-inducedhyperalgesia. (G. Bennett et al., Nerve growth factor inducedhyperalgesia in the rat hind paw is dependent on circulatingneutrophils, Pain 77(3)315-22 [1998]; see also E. Feher et al., Directmorphological evidence of neuroimmunomodulation in colonic mucosa ofpatients with Crohn's disease, Neuroimmunomodulation 4(5-6):250-57[1997]).

Visceral hyperalgesia, or pain hypersensitivity, is a common clinicalobservation in small intestinal bacterial overgrowth (SIBO), Crohn'sdisease, chronic pelvic pain syndrome, and irritable bowel syndrome(IBS). As many as 60% of subjects with IBS have reduced sensorythresholds for rectal distension compared to normal subjects. (H. Mertzet al., Altered rectal perception is a biological marker of patientswith the irritable bowel syndrome, Gastroenterol 109:40-52 [1995]).While the experience of pain is intertwined with a person's emotions,memory, culture, and psychosocial situation (D. A. Drossman and W. G.Thompson, Irritable bowel syndrome: a graduated multicomponent treatmentapproach, Ann. Intern. Med. 116:1009-16 [1992]) and the etiology forthis hyperalgesia has remained elusive, evidence shows that certaincytokine mediated-immune responses can influence the perception of pain.Cytokines, including IL-1(α and β), IL-2, IL-6, and TNF-α, can bereleased in response to a variety of irritants and can modulate theperception of pain, possibly through the mediation of kinin B₁ and/or B₂receptors (see, M. M. Campos et al., Expression of B ₁ kinin receptorsmediating paw oedema formalin-induced nociception. Modulation byglucocorticoids, Can. J. Physiol. Pharmacol. 73:812-19 [1995] R. O. P.de Campos et al., Systemic treatment with Mycobacterium bovis bacilluscalmest-guerin (BCG) potentiates kinin B, receptor agonist-inducednociception and oedema formation in the formalin test in mice,Neuropeptides 32(5):393-403 [1998]). Cytokine and neuropeptide levelsare altered in IBS. An increase in substance P (neuropeptide)-sensitivenerve endings has been observed in subjects with IBS. (X. Pang et al.,Mast cell substance P-positive nerve involvement in a patient with bothirritable bowel syndrome and interstitial cystitis, Urology 47:436-38[1996]). It has also been hypothesized that there is a sensitization ofafferent pathways in IBS. (E. A. Mayer et al., Basic and clinicalaspects of visceral hyperalgesia, Gastroenterol 1994; 107:271-93 [1994];L. Bueno et al., Mediators and pharmacology of visceral sensitivity:from basic to clinical investigations, Gastroenterol. 112:1714-43[1997]).

Fibromyalgia, typically involving global musculoskeletal and/orcutaneous pain, is, by definition; a hyperalgesic state since theAmerican College of Rheumatology defines fibromyalgia as a history ofglobal pain in the setting of 11 out of 18 predefined tender points. (F.Wolfe et al., The American College of Rheumatology 1990 criteria for theclassification of fibromyalgia, Arthritis Rheum. 33:160-72 [1990]).Evidence implies that the hyperalgesia of fibromyalgia is not simplytrigger point-related but rather a global hyperalgesia. (L. Vecchiet etal., Comparative sensory evaluation of parietal tissues in pairful andnonpainful areas in fibromyalgia and myofascial pain syndrome, In:Gebhart G F, Hammond D L, Jensen T S, editors, Progress in Pain Researchand Management, Vol. 2, Seattle. IASP Press, pp. 177-85 [1994]; Sorensenet al., Hyperexcitability in fibromyalgia, J. Rheumatol.15:152-55[1998]).

Cytokine and neuropeptide levels are altered in IBS, fibromyalgia, andCrohn's disease. It has been shown that levels of substance P, aneuropeptide associated with nociception, are elevated in thecerebrospinal fluid of subjects with fibromyalgia (H. Vaeroy et al.,Elevated CSF levels of substance P and high incidence of Raynaud'sphenomenon in patients with fibromyalgia: new features for diagnosis,Pain 32:21-26 [1988]; I. J. Russell et al., Elevated cerebrospinal fluidlevels of substance P in patients with the fibromyalgia syndrome,Arthritis Rheum. 37:1593-1601 [1994]). And an increase in substanceP-sensitive nerve endings has been observed in subjects with IBS andCrohn's disease, (X. Pang et al., Mast cell substance P-positive nerveinvolvement in a patient with both irritable bowel syndrome andinterstitial cystitis, Urology 47:436-38 [1996]; (C. R. Mantyh et al.,Receptor binding sites for substance P, but not substance K orneuromedin K, are expressed in high concentrations by arterioles,venules, and lymph nodules in surgical specimens obtained from patientswith ulcerative colitis and Crohn's disease, Proc. Natl. Acad. Sci.85:323-39 [1988]; S. Mazumdar and K. M. Das, Immumocytochemicallocalization of vasoctive intestinal peptide and substance P in thecolon from normal subjects and patients with inflammatory bowel disease,Am. J. Gastrol. 87:176-81 [1992]; C. R. Mantyh et al., Differentialexpression of substance P receptors in patients with Crohn's disease andulcerative colitis, Gastroenterol. 1995; 109:850-60 [1995]).

Patients with chronic pelvic pain are usually evaluated and treated bygynecologists, gastroenterologists, urologists, and internists, but inmany patients with chronic pelvic pain the examination and work-upremain unrevealing, and no specific cause of the pain, such asendometriosis, can be identified. In these cases the patient is commonlysaid to be suffering from a “chronic pelvic pain syndrome.” Once thediagnosis of chronic pelvic pain is made, treatment is typicallydirected to symptomatic pain management, rather than to an underlyingcause. (Wesselmann U, Czakanski P P, Pelvic pain: a chronic visceralpain syndrome, Curr. Pain Headache Rep. 5(1):13-9 [2001]).

Mental functioning and feelings of fatigue or depression can also beinfluenced by immune responses. Peripherally released pro-inflammatorycytokines, such as IL-1, IL-6 and TNF-α, act on brain cellular targetsand have been shown to depress spontaneous and learned behavior inanimals, the vagus nerve has been shown to mediate the transmissions ofthe immune message to the brain, resulting in production ofpro-inflammatory cytokines centrally in the brain. (R. Dantzer et al.,Cytokines and sickness behavior, Ann. N.Y. Acad. Sci. 840:586-90[1998]). In addition, there is bidirectional interplay betweenneurotransmitters and the immune system, lymphocytes and macrophagesbear surface receptors for the stress hormone corticotrophin releasinghormone (CRH), and they respond to CRH by enhanced lymphocyteproliferation and feedback upregulation of hypothalamic CRH production.(S. H. Murch [1998]).

Pituitary production of proopiomelanocortins, such as endorphins andenkephalins, is upregulated by IL-1 and IL-2, possibly Mediated by CRH,and lymphocytes and macrophages recognize these endogenous opiates viasurface receptors. (S. H. Murch [1998]). Lymphocytes (T_(H)2) andmacrophages also produce and process enkephalin to an active form.Macrophage-derived cytokines, such as TNF-α, IL-1, and IL-6, are knownto modulate neurotransmitter release and to affect overall neuralactivity, cytokines can induce classic illness behavior such assomnolence, apathy, depression, irritability, confusion, poor memory,impaired mental concentration, lever and anorexia.

While immunological responses of various severities can lead to symptomscharacteristic of irritable bowel syndrome, fibromyalgia, chronic pelvicpain syndrome, chronic fatigue syndrome, impaired mentation and/ormemory, depression, autism, ADHD, autoimmune diseases, and Crohn'sdisease, there has been a definite need to determine a causal factor,for each of these diagnostic categories, to which diagnostic testing andtreatment can be directed effectively.

SIBO has, until recently, mostly been suspected in subjects withsignificant malabsorptive sequelae. Most of the described cases of SIBOinvolve anatomic alterations such as physical obstruction (E. A. Deitchet al., Obstructed intestine as reservoir for systemic infection, Am. J.Surg. 159:394 [1990]), surgical changes (e.g., L. K. Enander et al., Theaerobic and anaerobic microflora of the gastric remnant more than 15years after Billroth II resection, Scand. J. Gastroenterol. 17:715-20[1982]); direct communication of the small intestine with coloniccontents such as fistulae (O. Bergesen et al., Is vitamin B12malabsorption in bile fistula rats due to bacterial overgrowth? A studyof bacterial metabolic activity in the small bowel, Scand. J.Gastroenterol. 23-471-6 [1988]) and ileocecal valve dysfunction(surgical or otherwise) (W. O. Griffin, Jr, et al., Prevention of smallbowel contamination by ileocecal valve, S. Med. J:64: 1056-8 [1971], P.Rutgeerts et al., Ileal dysfunction and bacterial overgrowth in patientswith Crohn's disease, Eur, J. Clin. Invest. 11:199-206 [1981]). Lesscommonly, SIBO has been associated with chronic pancreatitis (E. Tretpiand A. Ferrieri, Intestinal bacterial overgrowth during chronicpancreatitis, Curr. Med. Res. Opin. 15:47-52 [1999]), hypochlorhydria(e.g., S. P. Pereira et al., Drug-induced hypochlorhydria causes highduodenal bacterial counts in the elderly, Aliment Pharmacol. Ther.12:99-104 [1998]), and immunodeficiency (C. Pignata et al., Jejunalbacterial overgrowth and intestinal permeability in children withimmunodeficiency-syndromes, Gut 31:879-82 [1990], G. M. Smith et al.,Small intestinal bacterial overgrowth in patients with chroniclymphocytic leukemia, J. Clin. Pathol. 43:57-9 [1990]).

SIBO has been associated with infections of the abdominal cavity incases of alcoholic cirrhosis. (F. Casafont Morencos et al., Small bowelbacterial overgrowth in patients with alcoholic cirrhosis, Dig. Dis:Sci. 40(6):1252-1256 [1995]; J. Chesta et al., Abnormalities in proximalsmall bowel motility in patients with cirrhosis, Hepatology 17(5):828-32[1993]; C. S. Chang et al., Small intestine dysmotility and bacterialovergrowth in cirrhotic patients with spontaneous bacterial peritonitis,Hepatology 28(5):1187-90 [1998]). SIBO has also been associated withsymptoms of chronic diarrhea, anorexia or nausea in elderly patients,and the prevalence of overgrowth in subjects over 75 years old isreported to be as high as 79% even in the absence of clinically evidentclues of overgrowth or achlorhydria (S. M. Riordan et al., Smallintestinal bacterial overgrowth in the symptomatic elderly, Am. J.Gastroenterol. 92(1):47-51 [1997]). SIBO is also associated with chronicdigestive symptoms in children, especially infants under two years ofage (D. De Boissieu et al., Small-bowel bacterial overgrowth in childrenwith chronic digestive diarrhea, abdominal pain, or both, J. Pediatr:128(2):203-07 [1996]), and with chronic diarrhea after livertransplantation in children. (D. R. Mack et al., Small bowel bacterialovergrowth as a cause of chronic diarrhea after liver transplantation inchildren, Liver Transpl. Surg: 4(2): 166-69 [1998]).

Although diabetic enteropathy (F. Goldstein et al., Diabetic diarrheaand steatorrhea. Microbiologic and clinical observations, Ann. Intern.Med. 1970; 71:215-8 [1970]), idiopathic intestinal pseudo-obstruction(A. J. Pearson et al., Intestinal pseudo-obstruction with bacterialovergrowth in the small intestine, Am. J. Dig. Dis. 14:200-05 [1969])and scleroderma (I. J. Kahn et al., Malabsorption in intestinalscleroderma: Correction with antibiotics, N. Engl. J. Med. 274: 1339-44[1966]) are all known to produce motility disturbances leading to SIBO.Two previous reports have examined small bowel motility amonganatomically and medically naive SIBO subjects. (G. Vantrappen et al.,The interdigestive motor complex of normal subjects and patients withbacterial overgrowth of the small intestine, J. Clin. Invest. 59:1.158-66 [1977]; P. O. Stotzer et al., Interdigestive and postprandialmotility in small-intestinal bacterial overgrowth, Scand. J.Gastroenterol. 31:875-80 [1996]). These authors suggest that themajority of subjects with SIBO in the absence of other predisposingconditions, lack the phase III of interdigestive motility during shortterm recordings.

Phase III of interdigestive motility is a period of phasic contractionspropagating through the length of the small intestine, approximatelyonce every 87.2±5.4 minutes in the fasting state. (E. E. Soffer et al.,Prolonged ambulatory duodeno-jejunal manometry in humans: Normal valuesand gender effect, Am. J. Gastrol. 93:1318-23 [1998]). This fastingevent is responsible: for sweeping residue including small bowelcontaminants, such as accumulated bacteria, into the colon inpreparation for the next meal. (V. B. Nieuwenhujuijs et al., The role ofinterdigestive small bowel motility in the regulation of gut microflora,bacterial overgrowth, and bacterial translocation in rats, Ann. Surg.228: 188-93 [1998], E. Husebye, Gastrointestinal motility disorders andbacterial overgrowth, J. Intern. Med. 237:419-27 [1995]). The endogenouspeptide, motilin, is involved in the mediation of this event. (G.Vantrappen et al., Motilin and the interdigestive migrating motorcomplex in man, Dig. Dis. Sci. 24:497-500 [1979]). Other prokineticagents, such as erythromycin, are believed to act on the motilinreceptor and have been shown to rapidly induce an interdigestivemotility event in dogs and humans. (M. F. Otterson and S. K. Sarna,Gastrointestinal motor effect of erythromycin, Am. J. Physiol.259:G355-63; T. Tomomasa et al., Erythromycin induces migrating motorcomplex in human gastrointestinal tract, Dig. Dis. Sci. 31:157-61[1986]).

In general, the speed of transit through the small intestine is normallyregulated by inhibitory mechanisms located in the proximal and distalsmall intestine known as the jejunal brake and the ileal brake.Inhibitory feedback is activated to slow transit when end products ofdigestion make contact with nutrient sensors of the small intestine.(E.g., Lin, H. C., U.S. Pat. No. 5,977,175; Dobson, C. L. et al., Theeffect of oleic acid on the human ileal brake and its implications forsmall intestinal transit of tablet formulations, Pharm. Res. 16(1):92-96[1999]; Lin, H. C. et al., Intestinal transit is more potently inhibitedby fat in the distal (Ileal brake) than in the proximal (jejunal brake)gut, Dig. Dis. Sci. 42(1):19-25 [1997]; Lin, H. C. et al., Jejunalbrake: inhibition of intestinal transit by fat in the proximal smallintestine, Dig. Dis, Sci., 41(2):326-29 [1996a]).

Specifically, jejunal and ileal brakes slow transit by the release ofgut peptides such as peptide YY and by the activation of neural pathwayssuch as those involving endogenous opioids. (Lin, H. C. et al.,Fat-induced ileal brake in the dog depends on peptide YY, Gastroenterol.110(5):1491-95 [1996b]). Transit is then slowed by the stimulation ofnonpropagative intestinal contractions which inhibit movement of thelumenal content. The removal or impairment of these inhibitorymechanisms can lead to abnormally rapid transit. For example, inpatients with a history of resection of the terminal ileum, intestinaltransit can become uncontrolled and abnormally accelerated when theileal brake is no longer intact. Time for processing of food can then beso reduced that few end products of digestion are available to triggerthe jejunal brake as the remaining inhibitory mechanism.

Peptide YY and its analogs or agonists have been used to manipulateendocrine regulation of cell proliferation, nutrient transport, andintestinal water and electrolyte secretion. (E.g., Balasubramaniam,Analogs of peptide yy and uses thereof, U.S. Pat. No. 5,604,203;WO9820885A1; EP692971A1; Croom et al., Method of enhancing nutrientuptake, U.S. Pat. No. 5,912,227; Litvak, D. A. et al., Characterizationof two novel proabsorptive peptide YY analogs, BIM-43073D andBIM-43004C, Dig. Dis. Sci. 44(3):643-48 [1999]). A role for peptide YYin the regulation of intestinal motility, secretion, and blood flow hasalso been suggested, as well as its use in a treatment of malabsorptivedisorders (Liu, C. D. et al., Peptide YY: a potential proabsorbtivehormone for the treatment of malabsorptive disorders, Am. Surg.62(3):232-36 [1996]; Liu, C. D. et al., Intralumenal peptide YY inducescolonic absorption in vivo, Dis. Colon Rectum 40(4):478-82 [1997],Bilchik, A. J. et al., Peptide YY augments postprandial small intestinalabsorption in the conscious dog, Am. J. Surg. 167(6):570-74 [1994]).

Lin et al., immuno-neutralized peptide YY in vivo to block the ilealbrake response and, thus, showed that it is mediated by peptide YY.(Lin, H. C. et al., Fat-induced ileal brake in the dog depends onpeptide YY, Gastroenterology, 110(5):1491-95 [1996b]). Serum levels ofpeptide YY increase during the ileal brake response to nutrient infusioninto the distal ileum. (Spiller, R. C. et al., Further characterisationof the ‘ileal brake’ reflex in man—effect of ileal infusion of partialdigests of fat, protein, and starch on jejunal motility and release ofneurotensin, enteroglucagon, and peptide YY, Gut, 29(8):1042-51 [1988];Pironi, L. et al., Fat-induced ileal brake in humans: a dose-dependentphenomenon correlated to the plasma levels of peptide YY,Gastroenterology, 105(3):733-9 [1993]; Dreznik, Z. et al., Effect ofileal oleate on interdigestive intestinal motility of the dog, Dig. Dis.Sci., 39(7):1511-8 [1994]; Lin, C. D. et al., Interlumenal peptide YYinduces colonic absorption in vivo, Dis. Colon Rectum, 40(4):478-82[April 1997]). In contrast, in vitro studies have shown peptide YYinfused into isolated canine ileum dose-dependently increased phasiccircular muscle activity. (Fox-Threlkeld, J. A. et al., Peptide YYstimulates circular muscle contractions of the isolated perfused canineileum by inhibiting nitric oxide release and enhancing acetylcholinerelease, Peptides, 14(6):1171-78 [1993]).

Kreutter et al. taught the use of β₃-adrenoceptor agonists andantagonists for the treatment of intestinal motility disorders, as wellas depression, prostate disease and dyslipidemia (U.S. Pat. No.5,627,200).

Bagnol et al. reported the comparative immunovisualization of mu andkappa opioid receptors in the various cell layers of the ratgastrointestinal tract, including a comparatively large number of kappaopioid receptors in the myenteric plexus. (Bagnol, D. et al., Cellularlocalization and distribution of the cloned mu and kappa opioidreceptors in rat gastrointestinal tract, Neuroscience, 81(2):579-91[1997]). They suggested that opioid receptors can directly influenceneuronal activity in the gastrointestinal tract.

Kreek et al. taught the use of opioid receptor antagonists, such asnaloxone, naltrexone, and nalmefene, for the relief of gastrointestinaldysmotility. (Kreek et al., Method for controlling gastrointestinaldysmotility, U.S. Pat. No. 4,987,136). Riviere et al. taught the use ofthe opioid receptor antagonist fedotozine in the treatment of intestinalobstructions (Riviere, P. J. M. et al., U.S. Pat. No. 5,362,756).Opioid-related constipation, the most common chronic adverse effect ofopioid pain medications in patients who require long-term opioidadministration, such as patients with advanced cancer or participants inmethadone maintenance, has been treated with orally administeredmethylnaltrexone and naloxone. (Yuan, C. S. et al., Methylnaltrexone forreversal of constipation due to chronic methadone use: arandomizedcontrolled trial, JAMA 283(3):367-72 [2000]; Meissner, W. et al., Oralnaloxone reverses opioid associated constipation, Pain 84(1):105-9[2000]; Culpepper-Morgan, J. A., et al., Treatment of opioid-inducedconstipation with oral naloxone: a pilot study, Clin. Pharmacol. Ther.52(1): 90-95 [1992]; Yuan, C. S. et al., The safety and efficacy of oralmethylnaltrexone in preventing Morphine-induced delay in oral-cecaltransit time, Clin. Pharmacol. Ther. 61(4):467-75 [1997]; Santos, F. A.et al., Quinine-induced inhibition of gastrointestinal transit in mice:possible involvement of opioids, Eur. J. Pharmacol., 364(2-3):193-97[1999]. Naloxone was also reported to abolish the ileal brake in rats(Brown, N. J. et al., The effect of an opiate receptor antagonist theileal brake mechanism in the rat, Pharmacology; 47(4)230-36 [1993]).

Receptors for 5-hydroxytryptamine (5-HT) have been localized on variouscells of the gastrointestinal tract. (Gershon, M. D., Review article:roles played by 5-hydroxytryptamine in the physiology of the bowel,Aliment. Pharmacol. Ther., 13 Suppl 2:15-30 [1999]; Kirchgessner, A. L.et al., Identification of cells that express 5-hydroxytryptamine 1Areceptors in the nervous systems of the bowel and pancreas, J. Comp.Neurol., 15:364(3):439-455 [1-996]). Brown et al., reported thatsubcutaneous administration of 5-HT3 receptor antagonists, granisetronand ondansetron, in rats delayed intestinal transit of a baked bean mealbut abolished, the ileal brake induced by ileal infusion of lipid. Theypostulated the presence of 5-HT3 receptors on afferent nerves thatinitiate reflexes that both accelerate and delay intestinal transit.(Brown, N. J. et al., Granisetron and ondansetron: effects on the ilealbrake mechanism in the rat, J. Pharm. Pharmacol. 45(6):521-24 [1993]).Kuemmerle et al. reported neuro-endocrine 5-HT-mediation ofmotilin-induced accelerated gastrointestinal motility. (Kuemmerle, J. F.et al., Serotonin neural receptors mediate motilin-induced motility inisolated, vascularly perfused canine jejunum, J. Surg. Res., 45(4)357-62[1988]).

Ninety-five percent of the human body's stores of 5-hydroxytryptamine(5-HT), also known as serotonin, are found in the gastrointestinaltract. (Gershon, M. D., The Second Brain, New York: Harper Collins[1998]). In the intestines, the vast majority of 5-HT is located in theenterochromaffin (EC) cells of the mucosa (Gershon [1998]). 5-HT is alsoreleased by myenteric 5-HT neurons in the myenteric plexus. (Gershon, M.D., The enteric nervous system, Annu Rev Neurosci 4: 227-272 [1981];Gershon, M. D. et al., Serotonin: synthesis and release from themyenteric plexus of the mouse intestine, Science 149: 197-199 [1965];Holzer, P., and G. Skofitsch, Release of endogenous 5-hydroxytryptaminefrom the myenteric plexus of the guinea-pig isolated small intestine, BrJ Pharmacol 81: 381-386 [1984]; Penttila, A., Histochemical reactions ofthe enterochromaffin cells and the 5-hydroxytryptamine content of themammalian duodenum, Acta Physiol Scand Suppl 281: 1-77 [1966]). Theseintrinsic 5-HT neurons receive input from parasympathetic andsympathetic fibers (Gershon, M. D., and D. L. Sherman, Noradrenergicinnervation of serotoninergic neurons in the myenteric plexus, J CompNeurol 259: 193-210. [1987]) and provide input to the motor neurons intheir vicinity to suggest that they are interneurons. 5-HT3 receptorsare widely expressed by these myenteric 5-FIT neurons as well as theirneighboring neurons: (Galligan, J. J., Electrophysiological studies of5-hydroxytryptamine receptors on enteric neurons, Behav Brain Res 71:199-201 [1996]; Zhou, X, and J. J. Galligan, Synaptic activation andproperties of 5-hydroxytryptamine(3) receptors in myenteric neurons ofguinea pig intestine, J Pharmacol Exp Ther 290: 803-10 [1999]), However,the physiologic function of these myenteric 5-HT neurons is not known.(E. G. Gershon, M. D. Review article: roles played by5-hydroxytryptamine in the physiology of the bowel, Aliment PharmacolTher 13 Suppl 2: 15-30, 1999]; Grider, J. R. et al., 5-HT released bymucosal stimuli initiates peristalsis by activating 5-HT4/5-HT1preceptors on sensory CGRP neurons, Am J Physiol 270: G778-G782 [1996]).

Regardless of the source of 5-HT (mucosal vs. neuronal or both), thesignaling role of this molecule is facilitated by the availability of a5-HT reuptake transporter called SERT that terminates the signal withits removal. (Wade, P. R. et al., Localization and function of a 5-HTtransporter in crypt epithelia of the gastrointestinal tract, J Neurosci16: 2352-64 [1996]). Since-SERT is a part of the plasma membrane ofserotonergic neurons (Blakely, R. D. et al., Cloning and expression of afunctional serotonin transporter from rat brain, Nature 354: 66-70[1991]), these transporters are ideally positioned to remove neuronal5-HT after signaling is completed. Serotonergic nerves are, however,absent from the intestinal mucosa. (Furness, J. B., and M. Costas, Theenteric nervous system, New York Churchill Livingston [1987]). Instead,mucosal 5-HT from EC cells is removed by SERT expressed by neighboringepithelial cells. (Chen, J. X. et al., Guinea pig 5-HT transporter:cloning, expression, distribution, and function in intestinal sensoryreception, Am J Physiol 275: G433-G448 [1998]).

The action of SERT is blocked by drugs that inhibit the reuptaketransporter. These serotonin-selective reuptake inhibitors (SSRI) arewidely used as antidepressants. The most commonly prescribed example isfluoxetine (Prozac). These agents significantly alter the peristalticresponse. Wade et al. reported that fluoxetine initially accelerated thepassage of a pellet through an isolated segment of guinea pig colon tosuggest potentiation of the peristaltic effect of 5-HT when the removalof this molecule was inhibited (Wade et al. [1996]). However, as thedose of the SSRI was increased, the transit of the pellet became slowerand slower. This observation with fluoxetine suggested to Gershon that5-HT receptors became desensitized when an excess of 5-HT stayed aroundfor a longer period of time and traversed further away from its mucosalsource (Gershon [1998]). These are then the current concepts to explainthe common gastrointestinal side effects of SSRIs including nausea(excess 5-HT acting on extrinsic sensory nerves) and diarrhea (excess5-HT acting on intrinsic primary afferent neurons to initiateperistalsis; Gershon [1998]).

The current scientific foundation for understanding the role ofserotonin in normal and abnormal motility of the small intestine hasbeen based on the role of mucosal serotonin in two enteric functions.The first is as the neurotransmitter, via the activation of intrinsicprimary afferent neurons (IPAN), for the peristaltic reflex, whichmediates colonic evacuation, and for the mucosal secretory reflex.(E.g., Grider, J. R. et al., 5-Hydroxytryptamine4 receptor agonistsinitiate the peristaltic reflex in human, rat, and guinea pig intestine,Gastroenterology, 115(2):370-80 [1998]; Jin, J. G. et al., Propulsion inguinea pig colon induced by 5-hydroxytryptamine (HT) via 5-HT4 and 5-HT3receptors, J. Pharmacol. Exp. Ther., 288(1):9397 [1999]; Foxx-Orenstein,A. E. et al., 5-HT4 receptor agonists and delta-opioid receptorantagonists act synergistically to stimulate colonic propulsion, Am J.Physiol., 275(5 Pt. 1):G979-83 [1998]; Foxx-Orenstein, A. E., Distinct5-HT receptors mediate the peristaltic reflex induced by mucosal stimuliin human and guinea pig intestine, Gastroenterology 111(5):1281-90[1996]; Wade, P. R. et al., Localization and function of a 5-HTtransporter in crypt epithelia of the gastrointestinal tract; J.Neurosci., 16(7):2352-64 [1996]; Grinder, J., Gastrin-releasing peptide(GRP) neuron we excitatory neurons in the descending phase of theperistaltic reflex, Gastronenterology 116: A1000 [1999]; Cooke, H., M.Sidhu, and Y. Wang, 5-HT activates neural reflexes regulating secretionin the guinea pig colon, Neurogastroenterol Motil 9: 181-6 [1997];Cooke, H. J., and H. V. Carey, Pharmacological analysis of5-hydroxytryptamine actions on guinea-pig ileal mucosa, Eur J Pharmacol111: 329-37, [1985]; Frieling, T., J. Wood, and H. Cooke, Submucosalreflexes: distension-evoked ion transport in the guinea pig distalcolon, Am J Physiol 263: G91-96 [1992]; Hardcastle, J., and P.Hardcastle, Comparison of the intestinal secretory responses to5-hydroxytryptamine in the rat jejunum and ileum in-vitro, J PharmPharmacol 49: 1126-31 [1997]; Kinsman, R. I, and N. W. Read, Effect ofnaloxone on feedback regulation of small bowel transit by fat,Gastroenterology 87: 335-337 [1984]).

The second enteric role for 5-HT is as the signal to the brain aboutlumenal conditions, linking mucosal stimuli with the brain via extrinsicprimary sensory neurons. (Blackshaw, L. A, and D. Grundy, Effects of5-hydroxytryptamine on discharge of vagal mucosa afferent fibres fromthe upper gastrointestinal tract of the ferret, J Auton New Syst 45:41-50 [1993]). On the basis of this understanding, concepts have evolvedto explain the irritable bowel syndrome as a condition of serotoninexcess (leading to diarrhea from excessive peristalsis) (Gershon[1998]), even as the constipation typical of this syndrome remainspuzzling. Similar explanations have also been used to explain thediarrhea reported by patients taking SSRI (e.g. Prozac).

The intestinal response to 5-HT has previously been described in termsof the peristaltic reflex in in vitro models. Bulbring and Crema firstshowed that lumenal 5-HT resulted in peristalsis. (Bulbring et al., J.Physiol. 140:381-407 [1959]; Bulbring et al., Brit. J. Pharm. 13:444-457[1958]). Since the stimulation of peristalsis by 5-HT was unaffected byextrinsic denervation (Bulbring et al., QJ Exp. Physiol. 43:26-37[1958]), the peristaltic reflex was considered to be intrinsic to theenteric nervous system. Using a modified Trendelenburg model thatcompartmentalized the peristaltic reflex into the sensory limb, theascending contraction limb (orad to stimulus) and the descendingrelaxation limb (aboard to stimulus), Glider, et al. reported that (1)mucosal simulation but not muscle stretch released 5-HT to activate aprimary sensory neuron to release calcitonin gene-related peptide (CGRP)(Grider et al., Am. J. Physiol. 270:G778-G782 [1996]) via 5-HT4receptors in humans and rats (also 5-HT1p in rats) and 5-HT3 receptorsin guinea pigs; (2) cholinergic interneurons are then stimulated by CGRPto initiate both ascending contraction via an excitatory motor neuronthat depends on substances P and K and acetylcholine (Grider et al., Am.J. Physiol. 257:G709-G714 [1989]) and descending relaxation (Grider, Am.J. Physiol. 266:G1139-G1145 [1994]; Grider et al. [1996], Tin et al., J.Pharmacol. Exp: Ther. 288:93-97 [1999]) via an inhibitory motor neuronthat depends on pituitary adenylate cyclase-activating peptide (PACAP),nitric oxide and vasoactive inhibitory peptide (VIP) (Grider et al.,Neuroscience 54:521-526 [1993]; Grider et al., J. Auton. Nerv. Syst.50:151-159 [1:994]); and (3) peristalsis is controlled by [a] an opioidpathway that inhibits descending relaxation by suppressing the releaseof VIP; [b] a somatostatin pathway that inhibits this opioid pathway(Grider, Am. J. Physiol. 275:G973-G978 [1998]); and [c] a GABA (Grider,Am. J. Physiol. 267:G696-G701 [1994]) and a gastrin releasing peptide.(GRP) (Grider, Gastroenterol 116:A1000 [1999]) pathway that stimulateVIP release. An opioid pathway that inhibits the excitatory motorneurons responsible for ascending contraction has also been described(Gintzler et al., Br. J. Pharmacol. 75:199-205 [1982]; Yau et al., Am.J. Physiol. 250:G60-G63 [1986]). These observations are consistent withneuroanatomic and electrophysiological observations.

In addition, mucosal stroking has been found to induce 5-HT release byintestinal mucosal cells, which in turn activates a 5-HT4 receptor onenteric sensory neurons, evoking a neuronal reflex that stimulateschloride secretion (Kellum, J. M. et al., Stroking human jejunal mucosainduces 5-HT release and Cl⁻ secretion via afferent neurons and 5-HT4receptors, Am. J. Physiol. 277(3 Pt 1):G515-20 [1999]).

Agonists of 5-HT4/5, 5-HT3 receptors, as well as opioid Δ receptorantagonists, were reported to facilitate peristaltic propulsive activityin the colon in response to mechanical stroking, which causes theendogenous release of 5-HT and calcitonin gene-related protein (CGRP) inthe stroked mucosal area. (Steadman, C. J. et al., Selective5-hydroxytryptamine type 3 receptor antagonism with ondansetron astreatment for diarrhea-predominant irritable bowel syndrome: a pilotstudy, Mayo Clin. Proc. 67(0732.38 [1992]). Colonic distension alsoresults in CGRP secretion, which is associated with triggering theperistaltic reflex. 5-HT3 receptor antagonists have been used for thetreatment of autism. (E.g., Oakley et al., 5-HT3 receptor antagonistsfor the treatment of autism, U.S. Pat. No. 5,225,407).

Improved methods of detecting or diagnosing SIBO and SIBO-causedconditions are also a desideratum. Typically, detection of SIBO is doneby detecting hydrogen and/or methane exhaled in the breath. (E.g., P.Kerlin and L. Wong, Breath hydrogen testing in bacterial overgrowth ofthe small intestine, Gastroenterol 95(4):982-88 [1988]; A. Strocchi etal., Detection of malabsorption of law doses of carbohydrate: accuracyof various breath H ₂ criteria, Gastroenterol. 105(5):1404-1410 [1993];D. de Boissieu et al., [1996]; P. J. Lewindon et al., Bowel dysfunctionin cystic fibrosis: importance of breath testing, J. Paedatr. ChildHealth 34(1):79-82 [1998]). Hydrogen is a metabolic product of thefermentation of carbohydrates and amino acids by bacteria normally foundin the colon.

While the hydrogen that is produced in the colonic lumen may be excretedvia the lungs (exhaled breath) and the anus (flatus), these routes ofexcretion are responsible for the elimination of only a fraction of thetotal amount of hydrogen (10%) that is produced in the gut (Levitt, M.D. et al., Hydrogen (H ₂) catabolism in the colon of the rat, J Lab clinMed 84:163-167 [1974]).

The major mechanism for the removal of hydrogen produced by bacterialfermentation is the utilization of this gas by colonic bacteria thatcompetes to use hydrogen via one of three hydrogen disposal pathwaysthat are mutually exclusive. These pathways depend on the metabolism ofmethanogenic bacteria (Levitt, M. D. et al., H ₂ excretion afteringestion of complex carbohydrates, Gastroenterology 92:383-389 [1987]),acetogenic bacteria (Lajoie, R. et al., Acetate production from hydrogenand [c13] carbon dioxide by the microflora of human feces, Appl EnvironMicrobiol 54:2723-2727 [1988]) and sulfate-reducing bacteria (Gibson, G.R. et al., Occurrence of sulphate-reducing bacteria in human faeces andthe relationship of dissimilatory sulphate reduction to methanogenesisis the large gut, J Appl Bacteriol 65:103-111 [1988]). Methanogenicbacteria are more efficient than the other colonic bacteria in theelimination of lumenal hydrogen. (Strocchi, A. et al., Methanogensoutcompete sulphate reducing bacteria for H ₂ in the human colon, Gut35:1098-1101 [1994]). Acetogenic bacteria are uncommon, being found inthe intestinal populations of <5% of humans.

In the colon, sulfate-reducing bacteria reduces sulfate to hydrogensulfide. (MacFarlane, G. T. et al., Comparison of fermentation reactionsin different regions of the human colon, J Appl Bacteriol 72:57-64[1992]). Hydrogen sulfide is more damaging to tissues than anionicsulfide or sulfhydryl compounds. Intestinal bicarbonate facilitates theconversion of hydrogen sulfide produced by sulfate-reducing bacteria inthe gut to anionic sulfide. (Hamilton W A: Biocorrosion: The action ofsulphate-reducing bacteria, in Biochemistry of Microbial Degradation, C.Ratlidge (ed.) Dordrecht, Kluwer Academic Publishers, pages 555-570[1994]). Since sulfate-reducing bacteria are more common in patientswith the diagnosis of ulcerative colitis (Pitcher, M. C. L. et al.,Incidence and activities of sulphate-reducing bacteria in gut contentsof healthy subjects and patients with ulcerative colitis, FEMS MicrobiolEcol 86:103-112 [1991]), sulfate-reduding bacteria have been consideredfor a possible role in the pathogenesis of ulcerative colitis. (Florin,R. H. J. et al., A role for sulfate reducing bacteria in ulcerativecolitis?, Gastroenterology 98:A170 [1990]). This link has beenpostulated to be related to the injurious effect of hydrogen sulfide inimpairing the use of short chain fatty acids as fuel by colonicepithelial cells. (Roediger, W. E. W. et al., Sulphide impairment ofsubstrate oxidation in rat colonocytes: a biochemical basis forulcerative Colitis?, Clin Sci. 85:623-627 [1993]; Roediger, W. E. etal., Reducing sulfur-compounds of the colon impair colonocyte nutrition:implication of ulcerative colitis, Gastroenterology 1993, 104: 02-809).

Currently, clinical detection of sulfur-Containing gases is limited tothe detection of halitosis or bad breath. (Rosenberg, M. et al,Reproducibility and sensitivity of oral malodor measurements with aprotable sulphide monitor, J Dent Res. 1991 November; 70(11):1436-40).After garlic ingestion, the presence of allyl methyl sulfidedifferentiates the intestine rather than the mouth as the source of thesulfur-containing volatile gas (Suarez, F. et al., Differentiation ofmouth versus gut as site of origin of odoriferous breath gases aftergarlic ingestion, Am J Physiol 276(2 pt 1):G425-30 [1999]).

The role of sulfate-reducing bacteria in small intestinal bacterialovergrowth has not been studied, and the presence of sulfate-reducingbacteria are not detected using the standard breath testing method whichtypically detects only the presence of hydrogen, methane and carbondioxide.

There remains a need for an underlying causal factor, to whichdiagnostic testing and treatment can be directed, for SIBO andSIBO-caused conditions, such as irritable bowel syndrome, fibromyalgia;chronic pelvic pain syndrome; chronic fatigue syndrome autism;depression; impaired mentation, and/or memory; sugar craving; ADHD, MS,SLE and other autoimmune diseases; and Crohn's disease. This and otherbenefits of the present invention are described herein.

SUMMARY OF THE INVENTION

The present invention relates to the diagnosis or treatment of smallintestinal bacterial overgrowth (SIBO) and SIBO-caused conditions:SIBO-caused conditions, as described herein, include irritable bowelsyndrome (IBS), Crohn's disease (CD), fibromyalgia (FM), chronic pelvicpain syndrome (CPPS), chronic fatigue Syndrome (CFS), depression,impaired mentation, impaired memory, halitosis, tinnitus, sugar craving,autism, attention deficit/hyperactivity disorder (ADHD), drugsensitivity, and autoimmune diseases, for example, multiple sclerosis(MS), systemic lupus erythematosus (SLE).

In particular, the present invention relates to a method of treatingsmall intestinal bacterial overgrowth (SIBO) or a SIBO-caused conditionin a human subject. The method involves detecting in the subject by anysuitable detection means, the presence or absence of SIBO in thesubject. If SIBO is detected in the subject, the method further involvesdepriving the bacterial population, which constitutes the overgrowth inthe small intestine, of nutrient(s), sufficiently to inhibit the furthergrowth of the bacteria in the small intestine. With the growth of thebacteria constituting the SIBO condition thus inhibited, SIBO is atleast partially eradicated, as the subject's phase III interdigestivemotility is better able to clear the small intestine of the overgrowthand sweep the bacteria into the colon for eventual elimination from thebody. In addition, the at least partial eradication of the SIBOcondition also decreases the occurrence or magnitude of bacteria-relatedtoxicity, sepsis (in more severe or advanced SIBO), and/or the subject'sown immune responses, which ate continually triggered by the presence ofSIBO in non-immunocompromised subjects. The clinical symptoms of thesubject associated with SIBO or the SIBO-caused condition are,consequently, ameliorated by the at least partial eradication of SIBO.

In an alternative aspect of the present invention, the method involvesinhibiting the growth of the bacteria in the subject's small intestine,which bacteria constitute a SIBO condition that has been detected, byintroducing into the lumen of the small intestine, a pharmaceuticallyacceptable disinfectant or antibiotic composition in an amountsufficient to inhibit the growth of the bacteria, thereby at leastpartially eradicating SIBO in the human subject.

In still another alternative aspect of the present invention, the methodof treating small intestinal bacterial overgrowth (SIBO), or aSIBO-caused condition, in a human subject involves administering to thesubjects a pharmaceutically acceptable composition comprising astabilizer of mast cell membranes in the lumenal wall, in an amountsufficient to inhibit a mast cell-mediated immune response to SIBO inthe human subject.

The present invention also relates to a method of screening for theabnormally likely presence of SIBO in a human subject. The methodinvolves obtaining a serum sample from the subject, and thenquantitatively determining a concentration in the serum sample ofserotonin, one or more unconjugated bile acid(s), and/or folate. Anabnormally elevated serum concentration of one or more of thesesubstances is indicative of a higher than normal probability that SIBOis present in the subject. Thus, if the method of screening for thepresence of SIBO is employed as part of a blood work-up, either as partof a routine physical or by way of investigating a particular clinicalcomplaint of the subject's, the practitioner can be made aware that SIBOis more than normally likely to be present. The practitioner can thenelect to pursue a less convenient, but more diagnostically powerful,detection means for SIBO.

The present invention also relates to such a diagnostically powerfulSIBO detection means. In particular, this inventive method of detectingsmall intestinal bacterial overgrowth in a human subject involvesdetecting the relative amounts of methane, hydrogen, and at least onesulfur-containing gas in a gas mixture exhaled by the human subject,after the subject has ingested a controlled quantity of a substrate. Theexhaled gas mixture is at least partially produced by the metabolicactivity of the intestinal microflora of the subject.

The present invention is also directed to a method of determining therelative severity of SIBO or a SIBO-caused condition in a human subjectin whom SIBO has been detected. The method involves detecting in thesubject by suitable detection means, the presence or absence of SIBO,and, if the presence of SIBO is detected in the subject, the methodfurther involves detecting in the subject by suitable detection means arelative level of intestinal permeability, abnormally high intestinalpermeability indicating a relatively severe SIBO or SIBO-causedcondition in the subject.

The present invention also relates to a kit for the diagnosis of SIBO ora SIBO-caused condition, comprising: at least one breath samplingcontainer, a pre-measured amount of a substrate, and instructions for auser in detecting the presence or absence of SIBO by determining therelative amounts of methane, hydrogen, and at least onesulfur-containing gas in a gas mixture exhaled by the subject, afteringestion of a controlled quantity of the substrate. Thus, the kit isparticularly useful in practicing the inventive method of detectingsmall intestinal bacterial overgrowth in a human subject.

The present invention is further described by the disclosures of relatedU.S. applications: U.S. patent application Ser. No. 09/374,142, filed onAug. 11, 1999 and issued as U.S. Pat. No. 6,562,629 on May 13, 2003;U.S. patent application Ser. No. 09/546,119, filed on Apr. 10, 2000 andissued as U.S. Pat. No. 6,558,708 on May 6, 2003; U.S. patentapplication Ser. No. 09/420,046, filed on Oct. 18, 1999; U.S. patentapplication Ser. No. 09/359,583, filed on Jul. 22, 1999; U.S. patentapplication Ser. No. 08/832,307, filed on Apr. 3, 1997 and issued asU.S. Pat. No. 5,977,175 on Nov. 2, 1999; and U.S. patent applicationSer. No. 08/442,843, filed on[.] May 17, 1995, which are allincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows visual analog scores reported by subjects IBS and SIBObefore and after antibiotic treatment.

FIG. 2 shows visual analog scores from subjects with IBS and SIBO in apilot study, before and after antibiotic treatment.

FIG. 3 shows visual analog scores reported by subjects with fibromyalgiaand SIBO before and after antibiotic treatment.

FIG. 4 shows the correlation between the degree of improvement insymptoms and residual breath hydrogen production after antibiotictreatment in subjects with fibromyalgia and SIBO.

FIG. 5 shows visual analog scores reported by subjects with Crohn'sdisease and SIBO before and after antibiotic treatment.

FIG. 6 shows the correlation between degree of improvement in symptomsand residual breath hydrogen production after antibiotic treatment insubjects with Crohn's disease.

FIG. 7 shows that the severity of diarrheal symptoms is comparativelyless in SIBO patients who excrete methane.

FIG. 8 shows a typical effect of total enteral nutrition (TEN) regimenin the eradication of SIBO as detected by LBHT. In FIG. 8A(pre-treatment), SIBO was initially detected. After 14 days of the TENregimen, follow-up LBHT shows that SIBO had been at least partiallyeradicated (FIG. 8B).

FIG. 9 demonstrates that slowing of the rate of intestinal transit byfat depends on peptide YY (PYY), which is a physiological fat signalmolecule.

FIG. 10 demonstrates that demonstrates that slowing of the rate ofintestinal transit by fat depends on a serotonergic pathway.

FIG. 11 illustrates that the fat induced ileal brake depends on anondansetron-sensitive, efferent serotonergic 5-HT3-mediated pathway.

FIG. 12 shows that ondansetron abolishes the fat-induced ileal brake ina dose-dependent fashion.

FIG. 13 shows that ondansetron abolishes the fat-induced ileal brakewhen administered luminally but not intravenously.

FIG. 14 illustrates that the slowing of intestinal transit by distal gut5-HT depends on an ondansetron-sensitive 5-HT-mediated pathway in theproximal (efferent) and distal (afferent) gut.

FIG. 15 shows that lumenal 5-HT, delivered to the proximal gut, slowsintestinal transit in a dose-dependent fashion.

FIG. 16 illustrates that lumenal 5-HT slows intestinal transit viaactivation of an intestino-intestinal reflex.

FIG. 17 illustrates that slowing of intestinal transit by distal gut fatdepends on an extrinsic adrenergic neural pathway.

FIG. 18 illustrates that slowing of intestinal transit by PYY depends onan extrinsic adrenergic neural pathway.

FIG. 19 illustrates that slowing of intestinal transit by 5-HT in thedistal gut depends on a propranolol-sensitive extrinsic adrenergicneural pathway.

FIG. 20 illustrates that intestinal transit is slowed by norepinephrine(NE) in a 5-FIT-mediated neural pathway.

FIG. 21 illustrates that the fat-induced jejunal brake depends on theslowing effect of a naloxone-sensitive, opioid neural pathway.

FIG. 22 illustrates that the fat-induced ileal brake depends on theslowing effect of an efferent, naloxone-sensitive, opioid neuralpathway.

FIG. 23 shows that slowing of intestinal transit by distal gut 5-HTdepends on a naloxone-sensitive, opioid neural pathway.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to a method of treating smallintestinal bacterial overgrowth (SIBO) or a SIBO-caused condition in ahuman subject, including a juvenile or adult, of any age or sex.

The upper gastrointestinal tract of a human subject includes the entirealimentary canal, except the cecum, colon, rectum, and anus. While somedigestive processes, such as starch hydrolysis, begin in the mouth andesophagus, of particular importance as sites of digestion are thestomach and small intestine (or “small bowel”). The small intestineincludes the duodenum, jejunum, and the ileum. As the term is commonlyused in the art, the proximal segment of the small bowel, or proximalgut, comprises approximately the first half of the small intestine fromthe pylorus to the mid-gut. The distal segment, or distal gut includesapproximately the second half, from the mid-gut to the ileal-cecalvalve.

As used herein, “digestion” encompasses the process of breaking downlarge biological molecules into their smaller component molecules, forexample, proteins into amino acids. “Predigested” means that the processof digestion has already begun or has occurred prior to arrival in theupper gastrointestinal tract.

As used herein, “absorption” encompasses the transport of a substancefrom the intestinal lumen through the barrier of the mucosal epithelialcells into the blood and/or lymphatic systems.

Small intestinal bacterial overgrowth (SIBO), is an abnormal conditionin which aerobic and anaerobic enteric bacteria from the colonproliferate in the small intestine, which is normally relatively free ofbacterial contamination. SIBO is defined as greater than 10⁶ CFU/mLsmall intestinal effluent. (R. M. Donaldson, Jr., Normal bacterialpopulations of the intestine and their relation to intestinal function,N. Engl. J. Med. 270:938-45 [1964]).

Typically, the symptoms of SIBO include abdominal pain, bloating, gasand alteration in bowel habits, such as constipation and diarrhea.SIBO-caused conditions is used herein interchangeably with the term“SIBO-related conditions,” and regardless of ultimate causation, is acondition associated with the presence of SIBO in the subject.SIBO-caused conditions include other common symptoms, such as halitosis(“bad breath”), tinnitus (the experience of noise in the ears, such asringing, buzzing, roaring, or clicking, which may not be associated withexternally produced sounds), sugar craving, i.e., an intense desire forsweet foods or flavors, which can result in abnormally large consumptionof sweet foods and beverages and frequently leads to health-threateningobesity. Drug sensitivity is another common SIBO-caused condition, inwhich the subject is hypersensitive to medications, such asnon-steroidal anti-inflammatory medications, anti-insomniacs,antibiotics, or analgesics, and can suffer an unpredictableallergic-type reaction to medications at dose's that normally do notadversely affect the vast majority of patients. It is a benefit providedby the present invention that it provides a useful solution in thepresent tense, for many patients, to the problem of drug sensitivity,without requiring complex pharmacogenetic research and customized drugdevelopment.

Other SIBO-caused conditions, as described herein, can include thosefalling in the diagnostic categories of irritable bowel syndrome,Crohn's disease, fibromyalgia, chronic pelvic pain syndrome, chronicfatigue syndrome, depression, impaired mentation (including impairmentof the ability to concentrate, calculate, compose, reason, and/or useforesight or deliberate judgment), impaired memory, autism, attentiondeficit/hyperactivity disorder, and/or autoimmune diseases, such assystemic lupus erythematosus (SLE) or multiple sclerosis (MS).

In accordance with the invention, the SIBO-caused condition can be, butneed not be, previously diagnosed or suspected. The skilled medicalpractitioner is aware of suitable up-to-date diagnostic criteria bywhich a suspected diagnosis is reached. These diagnostic criteria arebased on a presentation of symptom(s) by a human subject. For example,these criteria include, but are not limited to, the Rome criteria forIBS (W. G. Thompson, Irritable bowel syndrome: pathogenesis andmanagement, Lancet 341:1569-72 [1993]) and the criteria for CFSestablished by the Centers for Disease Control and Prevention (CDC). (K.Fukuda et al., The chronic fatigue syndrome: a comprehensive approach toits definition and study, Ann. Intern. Med. 121:953-59 [1994]). Thediagnostic criteria for fibromyalgia of the American College ofRheumatology will also be familiar (F. Wolfe et al., The AmericanCollege of Rheumatology 1990 Criteria for the Classification ofFibromyalgia: Report of the Multicenter Criteria Committee, ArthritisRheum. 33:160-72 [1990]), as will be the criteria for depression or ADHDprovided for example, by the Diagnostic and Statistical Manual (DSM)-IVor its current version. (E.g., G. Tripp et al., DSM-IV ICD-10: acomparison of the correlates of ADHD and hyperkinetic disorder, J. Am.Acad. Child Adolesc. Psychiatry 38(2):156-64 [1999]) Symptoms ofsystemic lupus erythematosus include the 11 revised criteria of theAmerican College of Rheumatology, such as a typical malar or discoidrash, photosensitivity; oral ulcers, arthritis, serositis, or disordersof blood, kidney or nervous system. (E. M. Tan et al., The 1982 revisedcriteria for the classification of systemic lupus erythematosus [SLE],Arthritis Rheum. 25:1271-77 [1982]). Appropriate diagnostic criteria formultiple sclerosis are also familiar (e.g., L. A. Rolak, The diagnosisof multiple sclerosis, Neuronal Clin. 14(1):27-43 [1996]), as aresymptoms of Crohn's disease useful in reaching a suspected diagnosis.(e.g., J. M. Bozdech and R. G. Farmer, Diagnosis of Crohn's disease,Hepatogastroenterol. 37(1):8-17 [1990]; M. Tanaka and R. H. Riddell, Thepathological diagnosis and differential diagnosis of Crohn's disease,Hepatogastroenterol. 37(1):18-31 [1990]; A. B. Price and B. C. Morson,Inflammatory bowel disease: the surgical pathology of Crohn's diseaseand ulcerative colitis, Hum. Pathol. 6(1):7-29 [1975]). The practitioneris, of course not limited to these illustrative examples for diagnosticcriteria, but should use criteria that are current in the art.

Detection of the presence of SIBO in the human subject also corroboratesthe suspected diagnosis of the SIBO-caused condition, held by aqualified medical practitioner who, prior to the detection of SIBO inthe human subject, suspects from more limited clinical evidence that thehuman subject has, for example, irritable bowel syndrome, fibromyalgia,chronic fatigue syndrome, chronic pelvic pain syndrome, depression,autism, ADHD, an autoimmune disease, or Crohn's disease. By applying theinventive diagnostic method the suspected diagnosis is corroborated,i.e., confirmed, sustained, substantiated, supported, evidenced,strengthened, affirmed or made more firm.

The inventive method of treating SIBO, or a SIBO-caused condition,involves first detecting the presence or absence of SIBO in the subjectby suitable detection means. Detecting the presence or absence of SIBOis accomplished by any suitable means or method known in the art. Forexample, one preferred method of detecting SIBO is breath hydrogentesting. (E.g., P. Kerlin and L. Wong, Breath hydrogen testing inbacterial overgrowth of the small intestine, Gastroenterol. 95(4):982-88[1988]; A. Strocchi et al., Detection of malabsorption of low doses ofcarbohydrate: accuracy of various breath H ₂ criteria, Gastroenterol165(5):1404-1410 [1993]; D. de Boissieu et al., [1996], P. J. Lewindonet al., Bowel dysfunction in cystic fibrosis: importance of breathtesting, J. Paedatr. Child Health 34(1):79-82 [1998]). Breath hydrogenor breath methane tests are based on the fact that many obligately orfacultatively fermentative bacteria found in the gastrointestinal tractproduce detectable quantities of hydrogen or methane gas as fermentationproducts from a substrate consumed by the host, under certaincircumstances. Substrates include sugar such as lactulose, xylose,lactose, sucrose, or glucose. The hydrogen or methane produced in thesmall intestine then enters the blood stream of the host and aregradually exhaled.

Typically, after an overnight fast, the patient swallows a controlledquantity of a sugar, such as lactulose, xylose, lactose, or glucose; andbreath samples are taken at frequent time intervals, typically every 10to 15 minutes for a two- to four-hour period. Samples are analyzed bygas chromatography or by other suitable techniques, singly or incombination. Plots of breath hydrogen in patients with SIBO typicallyshow a double peak, i.e., a smaller early hydrogen peak followed by alarger hydrogen peak, but a single hydrogen peak is also a usefulindicator of SIBO, if peak breath hydrogen exceeds the normal range ofhydrogen for a particular testing protocol. (See, G. Mastropaolo and W.D. Rees, Evaluation of the hydrogen breath test in man: definition andelimination of the early hydrogen peak, Gut 28(6):721-25 [1987]).

A variable fraction of the population fails to exhale appreciablehydrogen gas during intestinal fermentation of lactulose; the intestinalmicroflora of these individuals instead produce more methane. (G.Corazza et al., Prevalence and consistency of low breath H ₂ excretionfollowing lactulose ingestion. Possible implications for the clinicaluse of the H ₂ breath test, Dig. Dis. Sci. 38(11):2010-16 [1993]; S. M.Riordan et al., The lactulose breath hydrogen test and small intestinalbacterial overgrowth; Am. J. Gastroenterol. 91(9); 1795-1803 [1996]).Consequently, in the event of an initial negative result for breathhydrogen, or as a precaution, methane and/or carbon dioxide contents ineach breath sample are optionally measured, as well as hydrogen, or asubstrate other than lactulose is optionally used. Also, acting as acheck, the presence of SIBO is demonstrated by a relative decrease inpeak hydrogen exhalation values for an individual subject afterantimicrobial treatment, in accordance with the present invention,compared to pretreatment values.

Another preferred method of detecting bacterial overgrowth is by gaschromatography with mass spectrometry and/or radiation detection tomeasure breath emissions of isotope-labeled carbon dioxide, methane, orhydrogen, after administering an isotope-labeled substrate that ismetabolizable by gastrointestinal bacteria but poorly digestible by thehuman host, such as lactulose, xylose, mannitol, or urea. (E.g., G. R.Swart and J. W. van den Berg, ¹³ C breath test in gastrointestinalpractice, Scand. J. Gastroenterol [Suppl.] 225:13-18 [1998]; S. F.Dellert et al., The 13C-xylose breath test for the diagnosis of smallbowel bacterial overgrowth in children, J. Pediatr. Gastroenterol. Nuts.25(2): 153-58 [1997]; C. E. King and P. P. Toskes, Breath tests in thediagnosis of small intestinal bacterial overgrowth, Crit. Rev. Lab. Sci.21(3):269-81 [1984]). A poorly digestible substrate is one for whichthere is a relative or absolute lack of capacity in a human forabsorption thereof or for enzymatic degradation or catabolism thereof.

Suitable isotopic labels include ¹³C or ¹⁴C. For measuring methane orcarbon dioxide, suitable isotopic labels can also include ²H and ³H or¹⁷O and ¹⁸O, as long as the substrate is synthesized with the isotopiclabel placed in a metabolically suitable location in the structure ofthe substrate, i.e., a location where enzymatic biodegradation byintestinal microflora results in the isotopic label being sequestered inthe gaseous product. If the isotopic label selected is a radioisotope,such as ¹⁴C, ³H, or ¹⁵O, breath samples can be analyzed bychromatography with suitable radiation detection means. (E.g., C. S.Chang et al., Increased accuracy of the carbon-14 D-xylose breath testin detecting small-intestinal bacterial overgrowth by correction withthe gastric emptying rate, Eur. J. Nucl. Med. 22(10):1118-22 [1995]; C.E. King and P. P. Toskes, Comparison of the 1-gram [ ¹⁴C]xylose, 10-gramlactulose-H ₂ ; and 80-gram glucose-H ₂ breath tests in patients withsmall intestine bacterial overgrowth, Gastroenterol. 91(6):1447-51[1986]; A. Schneider et al., Value of the ¹⁴ C-D-xylose breath test inpatients with intestinal bacterial overgrowth, Digestion 32(2):86-91[1985]).

Another preferred method of detecting small intestinal bacterialovergrowth is direct intestinal sampling from the human subject. Directsampling is done by intubation followed by scrape, biopsy, or aspirationof the contents of the intestinal lumen, including the lumen of theduodenum, jejunum, or ileum. The sampling is of any of the contents ofthe intestinal lumen including material of a cellular, fluid, fecal, orgaseous nature, or sampling is of the lumenal wall itself. Analysis ofthe sample to detect bacterial overgrowth is by conventionalmicrobiological techniques including microscopy, culturing, and/or cellnumeration techniques.

Another preferred method of detecting small intestinal bacterialovergrowth is by endoscopic visual inspection of the wall of theduodenum, jejunum, and/or ileum.

The preceding are merely illustrative and non-exhaustive examples ofmethods for detecting small intestinal bacterial overgrowth.

Another suitable, and most preferred, means for detecting the presenceor absence of SIBO is the present inventive method of detecting smallintestinal bacterial overgrowth in a human, subject, which involvesdetecting the relative amounts of methane, hydrogen, and at least onesulfur-containing gas in a gas mixture exhaled by said human subject,after the subject has ingested a controlled quantity of a substrate. Theinventive method of detecting small intestinal bacterial overgrowth ismore likely than conventional breath tests described above to detect thepresence of SIBO, because in some subjects a pattern exists that istermed “non-hydrogen, non-methane excretion” (see, e.g., Example 9chereinbelow). This pattern is the result of the subject having abacterial population constituting the SIBO condition, in which asulfate-reducing metabolic pathway predominates as the primary means forthe disposition of dihydrogen. In that condition, the removal of thehydrogen can be so complete that there is little residual hydrogen ormethane gas to be detected in the exhaled breath, compared to the amountof sulfur-containing gas, such as hydrogen sulfide or a volatilesulfhydryl compound detectable by the inventive method of detectingsmall intestinal bacterial overgrowth.

In accordance with the inventive method of detecting small intestinalbacterial overgrowth, the substrate is preferably a sugar, as describedhereinabove, and more preferably a poorly digestible sugar or anisotope-labeled sugar. The at least one sulfur-containing gas ismethanethiol, dimethylsulfide, dimethyl disulfide, an allyl methylsulfide, an allyl methyl sulfide, an allyl methyl disulfide, an allyldisulfide, an allyl mercaptan, or a methylmercaptan. Most preferably,the sulfur-containing gas is hydrogen sulfide or a sulfhydryl compound.

The detection or determination of the relative amounts of methane,hydrogen, and at least one sulfur-containing gas in the exhaled gasmixture is accomplished by means or systems known in the art, preferablyby means of gas chromatography Brunette, D. M. et al., The effects ofdentrifrice systems on oral malodor, J Clin Dent. 9:76-82 [1998];Tangerman, A. et al., A new sensitive assay for measuring volatilesulphur compounds in human breath by Tenax trapping and gaschromatography and its application in liver cirrhosis, Clin Chim Acta1983; May 9; 130(1):103-110 (1983)) and/or a radiation detection system,if appropriate. Most preferably, mass spectrometry is employed to detectthe relative amounts of methane, hydrogen, and at least onesulfur-containing gas in the exhaled gas mixture. (E.g., Spanel P, SmithD., Quantification of hydrogen sulphide in humid air by selected ionflow tube mass, spectrometry, Rapid Commun Mass Spectrom14(13):1:136-1140 [2000]). Combined gas chromatography and massspectrometry (GC/MS) is also useful. (E.g., Chinivasagam, H. N. et al.,Volatile components associated with bacterial spoilage of tropicalprawns, Int J Food Microbiol 1998 Jun. 30; 42(1-2):45-55). Mostpreferably, but not necessarily, the detection system employed requiresonly a single sample of exhaled gas mixture for the detection ofmethane, hydrogen, and at least one sulfur-containing gas. Detectionmethods that separately detect methane, hydrogen, and/or at least onesulfur containing gas are also useful.

Thus, thin-layer chromatography or high pressure liquid chromatographycan be useful for detection of volatile sulfur-containing compounds.(E.g., Tsiagbe, V. K. et al., Identification of volatile sulfurderivatives released from feathers of chicks fed diets with variouslevels of sulfur-containing amino acids, J Nutr 1987 117(11): 18859-65[1987]).

Direct-reading monitors for sulfides based on the use of anelectrochemical voltametric sensor or polarographic cell can also beemployed. Typically, gas is drawn into a sensor equipped with anelectrocatalytic sensing electrode. An electrical current is generated,by an electrochemical reaction proportional to the concentration of thegas. The quantity of the gas is typically determined by comparing to aknown standard.

In some embodiments of the inventive method of detecting SIBO in a humansubject, before detection, volatile sulfur-containing gases are trappedin Tenax absorbent (e.g., Tangerman, A. et al., Clin Chim Acta May 9;130(1):103-110 [1983]; Heida, H. et al., Occupational exposure andindoor air quality monitoring in a composting facility, Am Ind Hyg AssocJ 56(1):39-43[1995]) or other solvent/absorbent system such asdinitrophenyl thioethers (Tsiagbe, V. K. et al. [1987]).

It generally takes about 2 to 3 hours of the subjects's time and apre-test fast to accomplish breath testing for SIBO; thus, a quicker andmore convenient screening method to determine those subjects most likelyto have SIBO is desirable. Such a screening test allows the clinician tomake a more informed decision as to which patients would likely benefitfrom more definitive. SIBO testing, as described above. Thispre-screening reduces unnecessary inconvenience and expense for subjectswho are unlikely to have SIBO.

Hence, the present invention provides a method of screening for theabnormally likely presence of SIBO in a human subject. By abnormallylikely is meant a likelihood of SIBO greater than expected in thegeneral population. The inventive screening method involves obtaining aserum sample from the subject, which conventionally involves a blooddraw, followed by separation of the serum from the whole blood.Conventional immunochemical techniques, such as ELISA, employingcommercially available reagents, are used to quantitatively determine aconcentration in the serum sample of serotonin (5-HT), one or moreunconjugated bile acids (e.g., total bile acids or individual bileacids, e.g., deoxycholic acid), and/or folate, an abnormally elevatedserum concentration of one or more of these being indicative of a higherthan normal probability that SIBO is present in the subject. Suchquantitative immunochemical determinations of serum values are also madecommercially (e.g., Quest Diagnostics-Nichols Institute, 33608 OrtegaHighway, San Juan Capistrano, Calif. 92690).

For example, a normal range for serum 5-HT is up to about 0.5 nanogramsper milliliter. The normal range for total bile acids in serum is about4.0 to about 19.0 micromole per liter, and for deoxycholic acid thenormal is about 0:7 to about 7.7 micromoles per liter. Normal ranges forother unconjugated bile acids are also known. The normal range for serumfolate is about 2.6 to about 20.0 nanograms per, milliliter. Inaccordance with the inventive method of screening, subjects with atleast one serum value beyond the normal range are thus more thannormally likely to have SIBO present and are candidates for furtherdiagnostic SIBO detection procedures.

The present invention also relates to a method of determining therelative severity of SIBO or a SIBO-caused condition in a human subjectin whom SIBO has been detected by a suitable detection means, asdescribed herein above. If the presence of SIBO is detected in thesubject, then suitable detection means are employed to detected in thesubject a relative level of intestinal permeability, compared to normal.Abnormally high intestinal permeability indicates a relatively severeSIBO or SIBO-caused condition in the subject, which alerts the clinicianthat a more aggressive SIBO treatment regimen is desirable.

Techniques for detecting intestinal permeability and normal intestinalpermeability ranges are known. (E.g., Haase, A. M. et al., Dual sugarpermeability testing in diarrheal disease, J, Pediatr. 136(2):232-37[2000]; Spiller, R. C. et al., Increased rectal mucosal endocrine cells,T lymphocytes, and increased gut permeability following acuteCampylobacter enteritis and in post dysenteric irritable bowel syndrome,Gut 47(6):804-11 [2000]; Smecuol, E. et al., Sugar tests detect celiacdisease among first degree relatives, Am. J. Gastroenterol.94(12):3547-52 [1999]; Cox; M. A. et al., Measurement of smallintestinal permeability markers, lactulose and mannitol in serum:results disease, Dig. Dis. Sci. 44(2):402-06 [1999]; Cox, M. A. et. al.,Analytical method for the quantitation of mannitol and disaccharides inserum: a potentially useful technique in measuring small intestinalpermeability in vivo, Clin. Chim. Acta 263(2):197-205 [1997]; Fleming,S. C. et al., Measurement of sugar probes in serum: an alternative tourine measurement in intestinal permeability testing, Clin. Chem.42(3):445-48 [1996]).

Briefly, intestinal permeability is typically accomplished by measuringthe relative serum or urine levels of two sugars, after ingestion ofcontrolled amounts by the subject. One of the sugars, for examplemannitol, is chosen because it more typically more easily absorbedthrough the intestinal mucosa than the other sugar, for example,lactulose. Then about two hours after ingestion, a serum or urine sampleis taken, and the ratio of the two sugars is determined. The closer theratio of the two sugars in the sample approaches the ratio originallyingested, the more permeable is the subject's intestine.

After the presence of SIBO has been detected in the subject, inaccordance with the inventive method of treating small intestinalbacterial overgrowth (SIBO) or a SIBO-caused condition in a humansubject, the proliferating bacterial population constituting the SIBO isdeprived of nutrient(s) sufficiently to inhibit the growth of thebacteria in the small intestine, which results in at least partiallyeradicating SIBO in the human subject.

Depriving the bacterial population of nutrient(s) is accomplished by anyof a number of means.

For example, in some embodiments of the method of treating SIBO or aSIBO-caused condition, the subject consumes for a sustained period, adiet consisting essentially of nutrients that upon arrival in the uppergastrointestinal tract of the subject, are at least partiallypredigested. The sustained period being sufficient to at least partiallyeradicate SIBO in the human subject is at least about three days,preferably about 7 to about 18 days, and more preferably about 10 toabout 14 days.

In some embodiments or the method, the at least partially predigestednutrient(s) are contained in a commmestible total enteral nutrition(TEN) formulation, which is also called an “elemental diet.” Suchformulations are commercially available, for example, Vivonex® T.E.N.(Sandoz Nutrition, Minneapolis, Minn.) and its variants, or the like.(See; e.g., Example 11 hereinbelow). A useful total enteral nutritionformulatin satisfies all the subject's nutritional requirements,containing free amino acids, carbohydrates, lipids, and all essentialvitamins and minerals, but in a form that is readily absorbable in theupper gastrointestinal tract, thus depriving or “starving” the bacterialpopulation constituting the SIBO of nutrients of at least some of thenutrients they previously used for proliferating. Thus, bacterial growthin the small intestine is inhibited.

In another embodiment of the inventive method, a pancreatic enzymesupplement is administered to the subject before or substantiallysimultaneously with a meal, such that nutrients contained in the mealare at least partially predigested upon arrival in the uppergastrointestinal tract of the subject by the activity of the pancreaticenzyme supplement. Useful pancreatic enzyme supplements are commerciallyavailable, commonly called “Pancreatin”; such supplements containamylase, lipase, and/or protease. Representative methods ofadministering the pancreatic enzyme supplement include giving,providing, feeding or force-feeding, dispensing, inserting, injecting,infusing, prescribing, furnishing, treating with, taking, swallowing,ingesting, eating or applying.

In a preferred embodiment, depending on the formulation, the pancreaticenzyme supplement is administered up to a period of 24 hours prior toingestion of the food or nutrient comprising the meal, but mostpreferably between about 60 to 0 minutes before ingestion, which issubstantially simultaneously with the meal. The period of time prior toingestion is determined on the precise formulation of the composition.For example, a controlled release formulation can be administered longerbefore the meal. Other quick release formulations can be takensubstantially simultaneously with the meal.

In other embodiments of the method of treating small intestinalbacterial overgrowth or a SIBO-caused condition, depriving the bacterialpopulation of nutrient(s) involves enhancing the digestion and/orabsorption of the nutrient(s) in the upper gastrointestinal tract of thehuman subject by slowing transit of the nutrient(s) across the uppergastrointestinal tract of the human subject, thereby at least partiallydepriving the bacterial population of the nutrient(s). These embodimentsof the inventive take advantage of a novel understanding of theperipheral neural connections that exist between the enteric nervoussystem of the upper gastrointestinal tract, including an intrinsicserotonergic neural pathway, and the vertebral ganglia, and thence tothe central nervous system. The present invention provides a means toenhance region-to region (e.g., intestino-intestinal reflex)communications by way of replicating 5-HT as a signal (or releasing 5-HTat a distance as a surrogate signal). Thus, the present inventionprovides a way to increase 5-HT in locations in the central nervous bytransmitting a neural signal from the gut, or to transmit a5-HT-mediated neural signal originating in one location in the gut viaan intrinsic cholinergic afferent neural pathway to a second distantlocation in the gut where a serotonergic signal of the same or greaterintensity is replicated.

The present technology, therefore, allows neurally mediated modulationof the rate of upper gastrointestinal transit in the human subject. Thepresent invention allows the artificially directed transmission and/oramplification of nervous signals from one location in the entericnervous system to another via a prevertebral ganglion, bypassing thecentral nervous system. The invention takes advantage of an intrinsicserotonergic neural pathway involving an intrinsic cholinergic afferentneural pathway that projects from peptide YY-sensitive primary sensoryneurons in the intestinal wall to the prevertebral celiac ganglion. Theprevertebral celiac ganglion is in turn linked by multiple prevertebralganglionic pathways to the central nervous system, to the superiormesenteric ganglion, to the inferior mesenteric ganglion, and also backto the enteric nervous system via an adrenergic efferent neural pathwaythat projects from the prevertebral celiac ganglion to one or moreenterochromaffincells in the intestinal mucosa and to serotonergicinterneurons that are, in turn, linked in the myenteric plexus orsubmucous plexus to opioid interneurons. The opioid interneurons are inturn linked to excitatory and inhibitory motoneurons. The opioidinterneurons are also linked by an intestino-fugal opioid pathway thatprojects to the prevertebral celiac ganglion, with one or more neuralconnections therefrom to the central nervous system, including thespinal cord, brain, hypothalamus, and pituitary, and projecting backfrom the central nervous system to the enteric nervous system.

In particular, the present invention employs a method of manipulatingthe rate of upper gastrointestinal transit of food or nutrinetsubstance(s). The method involves administering by an oral or enteraldelivery route a pharmaceutically acceptable composition comprising anactive agent to the upper gastrointestinal tract. To slow the rate ofupper gastrointestinal transit, the active agent is an active lipid; aserotonin, serotonin agonist, or serotonin re-uptake inhibitor; peptideYY or a peptide YY functional analog; calcitonin gene-related peptide(CGRP) or a CGRP functional analog; an adrenergic agonist; an opioidagonist; or a combination of any of any of these, which is delivered inan amount and under conditions such that the cholinergic intestino-fugalpathway, at least one prevertebral ganglionic pathway, the adrenergicefferent neural pathway, the serotonergic interneuron and/or the opioidinterneuron are activated thereby. This results in the rate of uppergastrointestinal transit in the subject being slowed, which is the basisfor prolonging the residence time of orally or enterally administeredfood or nutrient substances, thus promoting or enhancing theirdissolution and/or absorption in the upper gastrointestinal tract.

The inventive pharmaceutically acceptable compositions limit thepresentation of a food or nutrient substance to the proximal region ofthe small intestine for absorption.

Depending on the desired results, useful active agents include, activelipids; serotonin, serotonin agonists, or serotonin re-uptakeinhibitors; peptide YY or peptide YY functional analogs; CGRP or CGRPfunctional analogs; adrenergic agonists; opioid agonists; or acombination of any of any of these; antagonists of serotonin receptors,peptide YY receptors, adrenoceptors, opioid receptors, CGRP receptors,or a combination of any of these. Also useful are antagonists ofserotonin receptors, peptide YY receptors, CGRP receptors; adrenoceptorsand/or opioid receptors.

Serotonin, or 5-hydroxytryptamine (5-HT) is preferably used at a dose ofabout 0.03 to about 0.1 mg/kg of body mass. 5-HT3 and 5-HT4 serotoninreceptor agonists are known and include HTF-919 and R-093877(Foxx-Orenstein, A. E. et al., Am. J. Physiol. 275(5 Pt 1):G979-83[1998]); prucalopride; 2-[1-(4-Piperonyl)piperazinyl]benzothiazole;1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-butyl-4-piperidinyl]-1-propanone;and1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-2-methylsulphonylamino)ethyl-4-piperidinyl]-1-propanone.Serotonin re-uptake inhibitors include Prozac or Zoloft.

Useful serotonin receptor antagonists include known antagonists of5-HT3, 5-HT1P, 5-HT1A, 5-HT2, and/or 5-HT4 receptors. Examples includeondansetron or granisetron, 5HT3 receptor antagonists (preferred doserange of about 0.04 to 5 mg/kg), deramciclane (Varga, G: et al., Effectof deramciclane, a new 5-HT receptor antagonist, oncholecystokinin-induced changes in rat gastrointestinal function, Eur.J. Pharmacol. 367(2-3)315-23 [1999]), or alosetron. 5-HT4 receptorantagonists are preferably used at a dose of about 0.05 to 500picomoles/kg. 5-HT4 receptor antagonists include1-Piperidinylethyl1H-indole-3-carboxylate (SB203186);1-[4-Amino-5-chloro-2-(3,5-dimethoxyphenyl)methyloxy]-3-[1-[2methylsulphonylamino]ethyl]piperidin-4-yl]propan-1-one(RS 39604); 3-(Piperidin-1-yl)propyl 4-amino-5-chloro-2-methoxybenzoate.

Peptide YY (PYY) and its functional analogs are preferably delivered ata dose of about 0.5 to about 500 picomoles/kg. PYY functional analogsinclude PYY (22-36), BIM-43004 (Liu, C D. et al., J. Surg. Res.59(1):80-84 [1995]), BIM-43073D, BIM-43004C (Litvak, D. A. et al., Dig.Dig. Sci. 44(3):643-48 [1999]). Other examples are also known in the art(e.g., β alasubramaniam, U.S. Pat. No. 5,604,203).

PYY receptor antagonists preferably include antagonists of Y4/PP1, Y5 orY5/PP2/Y2, and most preferably Y1 or Y2. (E.g., Croom et al., U.S. Pat.No. 5,912,227) Other examples include BIBP3226, CGP71683A (King, P. J.et al., J. Neurochem. 73(2):641-46 [1999]).

CGRP receptor antagonists include human CGRP(8-37) (e.g., Foxx-Orensteinet al., Gastroenterol. 111(5):1281-90 [1996]).

Useful adrenergic agonists include norepinephrine.

Adrenergic or adrenoceptor antagonists include β-adrenoceptorantagonists, including propranolol and atenolol. They are preferablyused at a dose of 0:05-2 mg/kg.

Opioid agonists include delta-acting opioid egotists (preferred doserange is 0.05-50 mg/kg, most preferred is 0.05-25 mg/kg); kappa-actingopioid agonists (preferred dose range is 0.005-100 microgram/kg);mu-acting opioid agonists (preferred dose range is 0.05-25microgram/kg); and episilon-acting agonists. Examples of useful opioidagonists include deltorphins (e.g., deltorphin II and analogues),enkephalins (e.g., [d-Ala(2), Gly-ol (5)]-enkephalin [DAMGO];[D-Pen(2,5)]-enkephalin [DPDPE]), dinorphins,trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl-]benzeneacetamidemethane sulfonate (U-50; 488H), morphine, codeine, endorphin, orβ-endorphin.

Opioid receptor antagonists include mu-acting opioid antagonists(preferably used at a dose range of 0.05-5 microgram/kg); kappa opioidreceptor antagonists (preferably used at a dose of 0.05-30 mg/kg); deltaopioid receptor antagonists (preferably used at a dose of 0.05-200microgram/kg); and epsilon opioid receptor antagonists. Examples ofuseful opioid receptor antagonists include naloxone, naltrexone,methylnaltrexone, nalmefene, H2186, H3116, or fedotozine, e.,(+)-1-1[3,4,5-trimethoxy)benzyloxymethyl]-1-phenyl-N,N-dimethylpropylamine.Other useful opioid receptor antagonists are known (e.g., Kreek et al.,U.S. Pat. No. 4,987,136).

The active agents bled above are not exhaustive but rather illustrativeexamples, and one skilled in the art is aware of other useful examples.

As used herein, “active lipid” encompasses a digested or substantiallydigested molecule having a structure and function substantially similarto a hydrolyzed end-product of fat digestion. Examples of hydrolyzed endproducts are molecules such as diglyceride, monoglyceride, glycerol, andmost preferably free fatty acids or salts thereof.

In a preferred embodiment, the active agent is an active lipidcomprising a saturated or unsaturated fatty acid. Fatty acidscontemplated by the invention include fatty acids having between 4 and24 carbon atoms (C4-C24).

Examples of fatty acids contemplated for use in the practice of thepresent invention include caprolic acid, caprulic acid, capric acid,lauric acid, myristic acid, oleic acid, palmitic acid, stearic acid,palmitoleic acid, linoleic acid, linolenic acid, trans-hexadecanoicacid, elaidic acid, columbinic acid, arachidonic acid, behenic acideicosenoic acid, erucic acid, bressidic acid, cetoleic acid, nervonicacid, Mead acid, arachidonic acid, timnodonic acid, clupanodonic acid,docosahexaenoic acid, and the like. In a preferred embodiment, theactive lipid comprises oleic acid.

Also preferred are active lipids in the form of pharmaceuticallyacceptable salts of hydrolyzed fats, including salts of fatty acids.Sodium or potassium salts are preferred, but salts formed with otherpharmaceutically acceptable cations are also useful. Useful examplesinclude sodium- or potassium salts of caprolate, caprulate, caprate,laurate, myristate, oleate, palmitate, stearate, palmitolate, linolate,linolenate, trans-hexadecanoate, elaidate, columbinate, arachidate,behenate, eicosenoate, erucate, bressidate, cetoleate, nervonate,arachidonate, timnodonate, clupanodonate, docosahexaenoate, and thelike. In a preferred embodiment, the active lipid comprises ad oleatesalt.

The active agents suitable for use with this invention are employed inwell dispersed form in a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” encompasses any of thestandard pharmaceutical carriers known to those of skill in the art. Forexample, one useful carrier is a commercially available emulsion,Ensure® but active lipids, such as oleate or oleic acid are alsodispersible in gravies, dressings, sauces or other comestible carriers.Dispersion can be accomplished in various ways. The first is that of asolution.

Lipids can be held in solution if the solution has the properties ofbile (i.e., solution of mixed micelles with bile salt added), or thesolution has the properties of a detergent (e.g., pH 9.6 carbonatebuffer) or a solvent (e.g., solution of Tween). The second is anemulsion which is a 2-phase system in which one liquid is dispersed inthe form of small globules throughout another liquid that is immisciblewith the first liquid (Swinyard and Lowenthal, “PharmaceuticalNecessities” REMINGTON'S PHARMACEUTICAL SCIENCES, 17th ed., A R Gennaro(Ed), Philadelphia College of Pharmacy and Science, 1985p. 1296). Thethird is a suspension with dispersed solids (e.g., microcrystallinesuspension). Additionally, any emulsifying and suspending agent that isacceptable for human consumption can be used as a vehicle for dispersionof the composition. For example, gum acacia, agar, sodium alginate,bentonite, carbomer, carboxymethylcellulose, carrageenan, powderedcellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol 9,oleyl alcohol, polyvinyl alcohol, povidone, propylene glycolmonostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol,tragacanth, xantham gum, chondrus, glycerin, trolamine, coconut oil,propylene glycol, thyl alcohol malt, and malt extract.

Any of these formulations, whether it is a solution, emulsion orsuspension containing the active agent, can be incorporated intocapsules, or a microsphere or particle. (coated or not) contained in acapsule.

The pharmaceutically acceptable compositions containing the activeagent, in accordance with the invention, is in a form suitable for oralor enteral use, for example, as tablets, troches, lozenges, aqueous oroily suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, syrups, elixirs or enteral formulas. Compositionsintended for oral use are prepared according to any method known to theart for the manufacture of pharmaceutical compositions. Compositions canalso be coated by the techniques described in the U.S. Pat. Nos.4,256,108; 4,160,452; and 4,265,874, to form osmotic therapeutic tabletsfor controlled release. Other techniques for controlled releasecompositions, such as those described in the U.S. Pat. Nos. 4,193,985;and 4,690,822; 4,572,833 can be used in the formulation of the inventivepharmaceutically acceptable compositions.

An effective amount of active lipid is any amount that effective to slowgastrointestinal transit and control presentation of a food or nutrientsubstance to a desired region of the small intestine. For example, aneffective amount of active lipid, as contemplated by the instantinvention, is any amount of active lipid that can trigger any or all ofthe following reflexes: intestine-lower esophageal sphincter (relaxationof LES); intestino-gastric feedback (inhibition of gastric emptying);intestino-intestinal feedback (ileo-jejunal feedback/ileal brake,jejuno-jejunal feedback/jejunal brake, intestino-CNS feedback (forexample, intensifying intestinal signalling of satiety);intestino-pancreatic feedback (control of exocrine enzyme output);intestino-biliary feedback (control of bile flow); intestino-mesentericblood flow feedback (for the control of mucosal hyperemia);intestino-colonic feedback (so called gastro-colonic reflex whereby thecolon contracts in response to nutrients in the proximal smallintestine).

Methods of administering are well known to those of skill in the art andinclude preferably oral administration and/or enteral administration.Representative methods of administering include giving, providing,feeding or force-feeding, dispensing, inserting, injecting, infusing,perfusing, prescribing, furnishing, treating with, taking, swallowing,eating or applying. Preferably the pharmaceutically acceptablecomposition comprising the active agent is administered in the settingof a meal, i.e., along with or substantially simultaneously with themeal, most preferably an hour or less before the meal. It is also usefulto administer the active agent in the fasted state, particularly if thepharmaceutical composition containing the active agent is formulated forlong acting or extended release. In some embodiments, such as theinventive method for manipulating post-prandial blood flow, thepharmaceutical composition is also usefully administered up to an hourafter a meal, and most preferably within one hour before or after themeal.

In order to stretch biologic activity so that one has a convenient,daily dosage regimen, the present invention contemplates that theinventive compositions can be administered prior to ingestion of thefood, nutrient and/or drug.

In a preferred embodiment, the inventive compositions (depending on theformulation) are administered up to a period of 24 hours prior toingestion of the food, nutrient and/or drug, but most preferably betweenabout 60 to 5 minutes before ingestion. The period of time prior toingestion is determined on the precise formulation of the composition.For example, if the formulation incorporates a controlled releasesystem, the duration of release and activation of the active lipid willdetermine the time for administration of the composition. Sustainedrelease formulation of the composition is useful to ensure that thefeedback effect is sustained.

In a preferred embodiment, the pharmaceutically acceptable compositionof the invention contains an active lipid and is administered in aload-dependent manner which ensures that the dispersion of active lipidis presented to the entire length of the small intestine. Administrationis in one or more doses such that the desired effect is produced. Insome preferred embodiments, the load of active lipid per dose is fromabout 0.5 grams to about 2.0 grams, but can range up to about 25 gramsper dose as needed. Generally, patients respond well to the mostpreferred amount of active lipid, which is in the range of about 1.6 to3.2 grams. For patients who fail to respond to this dose range, a dosebetween 6 and 8 grams is typically effective.

Sequential dosing is especially useful for patients with short bowelsyndrome or others with abnormally rapid intestinal transit times. Inthese patients, the first preprandial administration of the active lipidoccurs in a condition of uncontrolled intestinal transit that can failto permit optimal effectiveness of the active lipid. A second (or more)preprandial administration(s) timed about fifteen minutes after thefirst or previous administration and about fifteen minutes before themeal enhances the patient's control of intestinal lumenal contents andthe effectiveness of the active lipid in accordance with the inventivemethods. Normalization of nutrient absorption and bowel controlthroughout the day, including during the patient's extended sleepinghours, is best achieved by a dietary regimen of three major meals withabout five snacks interspersed between them, including importantly, apre-bedtime snack; administration of a dose of the inventive compositionshould occur before each meal or snack as described above.

Treatment with the inventive compositions in accordance with theinventive methods can be of singular occurrence or can be continuedindefinitely as needed. For example, patients deprived of food for anextended period (e.g., due to a surgical intervention or prolongedstarvation), upon the reintroduction of ingestible food, can benefitfrom administration of the inventive compositions before meals on atemporary basis to facilitate a nutrient adaptive response to normalfeeding. On the other hand some patients, for example those withsurgically altered intestinal tracts (e.g., ileal resection), canbenefit from continued pre-prandial-treatment in accordance with theinventive methods for an indefinite period. However, clinical experiencewith such patients for over six years has demonstrated that afterprolonged treatment there is at least a potential for an adaptivesensory feedback response that can allow them to discontinue treatmentfor a number of days without a recurrence of postprandial diarrhea orintestinal dumping.

The use of pharmaceutically acceptable compositions of the presentinvention in enteral feeding contemplates adding the compositiondirectly to the feeding formula. The composition can either becompounded as needed into the enteral formula when the rate of formuladelivery is known (i.e., add just enough composition to deliver the loadof active lipids). Alternatively, the composition of the invention canbe compounded at the factory so that the enteral formulas are producedhaving different concentrations of the composition and can be usedaccording to the rate of formula delivery (i.e., higher concentration ofcomposition for lower rate of delivery).

If the inventive composition were to be added to an enteral formula andthe formula is continuously delivered into the small intestine, thecomposition that is initially presented with the nutrient formula allowsslowing the transit of nutrients that are delivered later. Except forthe start of feeding when transit can be too rapid because theinhibitory feedback from the composition has yet to be fully activated,once equilibrium is established, it is no longer logistically an issueof delivering the composition as a premeal although the physiologicprinciple is still the same.

Before dietary fats can be absorbed, the motor activities of the smallintestine in the postprandial period must first move the output from thestomach to the appropriate absorptive sites of the small intestine. Toachieve the goal of optimizing the movement of a substance through thesmall intestine, the temporal and spatial patterns of intestinalmotility are specifically controlled by the nutrients of the lumenalcontent.

Without wishing to be bound by any theory, it is presently believed thatearly in gastric emptying, before inhibitory feedback is activated, theload of fat entering the small intestine can be variable and dependenton the load of fat in the meal. Thus, while exposure to fat can belimited to the proximal small bowel after a small load, a larger load,by overwhelming more proximal absorptive sites, can spill further alongthe small bowel to expose the distal small bowel to fat. Thus, theresponse of the duodenum to fat limit the spread of fat so that moreabsorption can be completed in the proximal small intestine and less inthe distal small intestine. Furthermore, since the speed of movement oflumenal fat must decrease when more fat enters the duodenum, in order toavoid steatorrhea, intestinal transit is inhibited in a load-dependentfashion by fat. This precise regulation of intestinal transit occurswhether the region of exposure to fat is confined to the proximal gut orextended to the distal gut.

In accordance with the present invention it has been observed thatinhibition of intestinal transit by fat depends on the load of fatentering the small intestine. More specifically, that intestinal transitis inhibited by fat in a load-dependent fashion whether the nutrient isconfined to the proximal segment of the small bowel or allowed access tothe whole gut.

As described above, the inventive technology can also operate bytransmitting to and replicating at a second location in the uppergastrointestinal tract a serotonergic neural signal originating at afirst location in the proximal or distal gut of a mammal. For example,the first location can be in the proximal gut and the second locationcan be elsewhere in the proximal gut or in the distal gut. Orconversely, the first location can be in the distal gut and the secondlocation can be elsewhere in the distal gut or in the proximal gut.

Employing this inventive technology to slow the rate of uppergastrointestinal transit, during and after a meal, nutrient absorptionin the upper gastrointestinal tract is enhanced, depriving bacterialpopulations in the lower small intestine of nutrients. In response toluminal fat in the proximal or distal gut, a serotonin (5-HT)-mediatedanti-peristaltic slowing response is normally present. Therefore, someembodiments of the method involve increasing 5-HT in the gut wall byadministering to the mammal and delivering to the proximal and/or distalgut, an active lipid, or serotonin, a serotonin, agonist, or a serotoninre-uptake inhibitor.

Alternatively, the active agent is PYY, or a PYY functional analog. PYYor the PYY analog activates the PYY-sensitive primary sensory neurons inresponse to fat or 5-HT. Since the predominant neurotransmitter of thePYY-sensitive primary sensory neurons is calcitonin gene-related peptide(CGRP), in another embodiment, CGRP or a CGRP functional analog is theactive agent.

In other embodiments the point of action is an adrenergic efferentneural pathway, which conducts neural signals from one or more of theceliac, superior mesenteric, and inferior mesenteric prevertebralganglia, back to the enteric nervous system. The active agent is anadrenergic receptor (i.e., adrenoceptor) agonist to activate neuralsignal transmission to the efferent limb of the anti-peristaltic reflexresponse to luminal fat.

Since adrenergic efferent neural pathway(s) from the prevertebralganglia to the enteric nervous: system stimulate serotonergicinterneurons, which in turn stimulate enteric opioid interneurons, inother embodiments of the method, the active agent is 5-HT, 5-HT receptoragonist, or a 5-HT re-uptake inhibitor to activate or enhance neuralsignal transmission at the level of the serotonergic interneurons.

Alternatively, the active agent is an opioid receptor agonist toactivate or enhance neural signal transmission via the opioidinterneurons.

In accordance with the invention, pharmaceutically acceptablecompositions containing the active agent can be in a form suitable fororal use, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, syrups, elixirs or enteral formulas. Compositions intended fororal use can be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions and such compositions cancontain one or more other agents selected from the group consisting of asweetening agent such as sucrose, lactose, or saccharin, flavoringagents, such as peppermint, oil of wintergreen or cherry, coloringagents and preserving agents in order to provide pharmaceuticallyelegant and palatable preparations. Tablets containing the activeingredient in admixture with non-toxic pharmaceutically acceptableexcipients can also be manufactured by known methods. The excipientsused can be, for example, (1) inert diluents such as calcium carbonate,lactose, calcium phosphate or sodium phosphate; (2) granulating anddisintegrating agents such as corn starch, potato starch or alginicacid; (3) binding agents such as gum tragacanth, corn starch, gelatin oracacia, and (4) lubricating agents such as magnesium stearate, stearicacid or talc. The tablets can be uncoated or they can be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed. They can also becoated by the techniques described in the U.S. Pat. Nos. 4,256,108;4,160,452; and 4,265,874, to form osmotic therapeutic tablets forcontrolled release. Other techniques for controlled releasecompositions, such as those described in the U.S. Pat. Nos. 4,193,985;and 4,690,822; 4,572,833 can be used in the formulation of the inventivepharmaceutically acceptable compositions.

In some cases, formulations for oral use can be in the form of hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin. They can also be in the form of soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, for example,peanut oil, liquid paraffin, or olive oil.

In one embodiment of the present invention, the pharmaceuticallyacceptable composition is an enterically coated or a sustained releaseform that permits intestinal transit to be slowed for a prolonged periodof time.

In an alternative aspect of the method of treating small intestinalbacterial overgrowth (SIBO) or a SIBO-caused condition in a humansubject, after the presence of SIBO is detected in the human subject bysuitable detection means, as described above, a pharmaceuticallyacceptable disinfectant composition is introduced into the lumen of thesmall intestine so as to contact the bacteria constituting the SIBOcondition. The disinfectant composition is introduced in an amountsufficient to inhibit the growth of the bacteria in the small intestine,thereby at least partially eradicating SIBO in the human subject.

Preferably, the pharmaceutically acceptable disinfectant compositionconsists essentially of hydrogen peroxide; a bismuth-containing compoundor salt; or an iodine-containing compound or salt. The pharmaceuticallyacceptable disinfectant (i.e., bacteriocidal) composition can alsocontain other non-bacteriocidal ingredients, such as any suitablepharmaceutically acceptable carrier, excipient, emulsant, solvent,colorant, flavorant, and/or buffer, as described hereinabove.Formulations for oral or enteral delivery are useful, as describedhereinabove with respect to known delivery modalities for active agents,e.g., tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsions, hard or soft capsules,syrups, elixirs or enteral formulas.

Embodiments of disinfectant or bacteriocidal compositions containinghydrogen peroxide are known for internal use in vertebrates (e.g.,Ultradyne, Ultra Bio-Logics Inc., Montreal, Canada). Preferably, anaqueous solution of about 1% to about 3% (v/v) hydrogen peroxide isintroduced orally or otherwise enterally to the lumen, most convenientlyby ingestion.

Embodiments of disinfectant or bacteriocidal compositions containingbismuth compounds or salts are also known, for example,bismuth-2-3-dimercaptopropanol (BisBAL), bismuth thiols (e.g.,bismuth-ethanedithiol), or bismuth-3,4-dimercaptotoluene (BisTOL), andin over the counter preparations, such as PeptoBizmol. (See, e.g.,Domenico, P. et al., Activity of Bismuth Thiols against Staphylococciand Staphylococcal biofilms, Antimicrob. Agents Chemother. 45(5):1417-21[2001]).

Embodiments of disinfectant or bacteriocidal compositions containingiodine compounds or salts are also known, for example, povidone-iodinesolutions.

In still another alternative aspect of the method of treating smallintestinal bacterial overgrowth (SIBO) or a SIBO-caused condition in ahuman subject, after the presence of SIBO is detected in the humansubject by suitable detection means, as described above, apharmaceutically acceptable composition is administered to the subject.The pharmaceutically acceptable composition contains a stabilizer ofmast cell membranes in the lumenal wall of the small intestine, in anamount sufficient to inhibit a mast cell-mediated immune response in thehuman subject. This embodiment is a relatively aggressive treatment andis most useful in more severe or advanced SIBO, for example, asconfirmed by high intestinal permeability in the subject (seehereinabove). Suitable mast cell stabilizers include oxatamide orchromoglycate (potassium or sodium salts preferred). (e.g., Pacor, M. L.et al., Controlled study of oxatomide vs disodium chromoglycate fortreating adverse reactions to food, Drugs Exp Clin Res 18(3):119-23[1992]; Stefanini, G. F. et al., Oral cromolyn sodium in comparison withelimination diet in the irritable bowel syndrome, diarrheic type,Multicenter Study of 428 patients, Scand. J. Gastroenterol. 30(6):535-41[1995]; Andre, F. et al., Digestive permeability to different-sizedmolecules and to sodium cromoglycate in food allergy, Allergy Proc.12(5):293-98 [1991]; Lunardi, C. et al., Double-blind cross-over trialof oral sodium cromoglycate in patients with irritable bowel syndromedue to food intolerance, Clin Exp Allergy 21(5):569-72 [1991]; Burks, A.W. et al., Double-blind placebo-controlled trial of oral cromolyn inchildren with atopic dermatitis and documented food hypersensitivity, J.Allergy Clin. Immunol. 81(2):417-23 [1988]).

After the SIBO condition is at least partially eradicated, typicallywithin a couple of weeks, there is an improvement in the symptom(s) ofirritable bowel syndrome, fibromyalgia, chronic fatigue syndrome,chronic pelvic pain syndrome, autism, impaired mentation, impairedmemory, depression, ADHD, an autoimmune disease, or Crohn's disease. Itis a benefit of the inventive treatment method that after treatment,subjects routinely report feeling better than they have felt in years.

The inventive method of treating small intestinal bacterial overgrowth(SIBO) or a SIBO-caused condition in a human subject, as describedabove, can be optionally combined, simultaneously or in sequence, withother suitable methods of at least partially eradicating smallintestinal bacterial overgrowth, such as the following.

For example, at least partially eradicating the bacterial overgrowth isaccomplished by administering an antimicrobial agent, including but notlimited to a natural, synthetic, or semi-synthetic antibiotic agent. Forexample, a course of antibiotics such as, but not limited to, neomycin,metronidazole, teicoplanin, doxycycline, tetracycline, ciprofloxacin,augmentin, cephalexin (e.g., Keflex), penicillin, ampicillin, kanamycin,rifamycin, rifaximin, or vancomycin, which may be administered orally,intravenously, or rectally. (R. K. Cleary [1998]; C. P. Kelly and J. T.LaMont, Clostridium difficile infection, Annu. Rev. Med. 49:375-90[1998]; C. M. Reinke and C. R. Messick, Update on Clostridiumdifficile-induced colitis, Part 2, Am. J. Hosp. Pharm. 51(15):1892-1901[1994]).

Alternatively, an antimicrobial chemotherapeutic agent, such as a 4- or5-aminosalicylate compound is used to at least partially eradicate theSIBO condition. These can be formulated for ingestive, colonic, ortopical non-systemic delivery systems or for any systemic deliverysystems. Commercially available preparations include4-(p)-aminosalicylic acid (i.e., 4-ASA or para-aminosalicylic acid) or4-(p)-aminosalicylate sodium salt (e.g., Nemasol-Sodium® or Tubasal®).5-Aminosalicylates have antimicrobial, as well as anti-inflammatoryproperties (H. Lin and M. Pimentel, Abstract G3452 at Digestive DiseaseWeek, 100^(th) Annual Meeting of the AGA, Orlando, Fla. [1999]), inuseful preparations including 5-aminosalicylic acid (i.e., 5-ASA,mesalamine, or mesalazine) and conjugated derivatives thereof, availablein various pharmaceutical preparations such as Asacol®, Rowasa®,Claversal®, Pentasa®, Salofalk®, Dipentum® (olsalazine), Azulfidine®(SAZ; sulphasalazine), ipsalazine, salicylazobenzoic acid, balsalazide,or conjugated bile acids, such as ursodeoxycholic acid-5-aminosalicylicacid, and others.

Another preferred method of at least partially eradicating smallintestinal bacterial overgrowth, particularly useful when a subject doesnot respond well to oral or intravenous antibiotics or otherantimicrobial agents alone, is administering an intestinal lavage orenema, for example, small bowel irrigation with a balanced hypertonicelectrolyte solution, such as Go-lytely or fleet phosphosodapreparations. The lavage or enema solution is optionally combined withone or more antibiotic(s) or other antimicrobial agent(s). (E.g., J. A.Vanderhoof et al., Treatment strategies for small bowel bacterialovergrowth in short bowel syndrome, J. Pediatr. Gastroenterol. Nutr.27(2):155-60 [1998])

Another preferred method of at least partially eradicating smallintestinal bacterial overgrowth employs a probiotic agent, for example,an inoculum of a lactic acid bacterium or bifidobacterium. (A. S. Naiduet al., Probiotic spectra of lactic acid bacteria, Crit. Rev. Food Sci.Nutr. 39(1):13-126 [1999]; J. A. Vanderhoof et al. [1998]; G. W.Tannock, Probiotic properties of lactic acid bacteria: plenty of scopefor R & D, Trends Biotechnol. 15(7):270-74 [1997]; S. Salminen et al.,Clinical uses of probiotics for stabilizing the gut mucosal barrier:successful strains and future challenges, Antonie Van Leeuwenhoek70(2-4):347-58 [1997]). The inoculum is delivered in a pharmaceuticallyacceptable ingestible formulation, such as in a capsule, or for somesubjects; consuming a food supplemented with the inoculum is effective,for example a milk, yogurt, cheese, meat or other fermentable foodpreparation. Useful probiotic agents include Bifidobacterium sp. orLactobacillus species or strains, e.g., L. acidophilus, L. rhamnosus, L.plantarum, L. reuteri, L. paracasei subsp. paracasei, or L. caseiShirota, (P. Kontula et al., The effect of lactose derivatives onintestinal lactic acid bacteria, J. Dairy Sci. 82(2):249-56 [1999]; M.Alander et al., The effect of probiotic strains on the microbiota of theSimulator of the Human Intestinal Microbial Ecosystem (SHIME), Int. J.Food. Microbiol. 46(1):71-79 [1999]; S. Spanhaak et al., The effect ofconsumption of milk fermented by Lactobacillus casei strain Shirota onthe intestinal microflora and immune parameters in humans, Eur. J. Clin.Nutr. 52(12):899-907 [1998]; W. P. Charteris et al., Antibioticsusceptibility of potentially probiotic Lactobacillus species, J. FoodProt. 61(12):1636-43 [1998]; B. W. Wolf et al., Safety and tolerance ofLactobacillus reuteri supplementation to a population infected with thehuman immunodeficiency virus, Food Chem. Toxicol. 36(12):1085-94 [1998];G. Gardiner et al., Development of a probiotic cheddar cheese containinghuman-derived Lactobacillus paracasei strains, Appl. Environ. Microbiol.64(6):2192-99 [1998]; T. Sameshima et al., Effect of intestinalLactobacillus starter cultures on the behaviour of Staphylococcus aureusin fermented sausage, Int. J. Food Microbiol. 41(1):1-7 [1998]).

Optionally, after at least partial eradication of small intestinalbacterial overgrowth, use of antimicrobial agents or probiotic agentscan be continued to prevent further development or relapse of SIBO.

Another preferred method of at least partially eradicating smallintestinal bacterial overgrowth is by normalizing or increasing phaseIII interdigestive intestinal motility between meals with any of severalmodalities to at least partially eradicate the bacterial overgrowth, forexample, by suitably modifying the subject's diet to increase smallintestinal motility to a normal level (e.g., by increasing dietaryfiber), or by administration of a chemical prokinetic agent to thesubject, including bile acid replacement therapy when this is indicatedby low or otherwise deficient bile acid production in the subject.

For purposes of the present invention, a prokinetic agent is anychemical that causes an increase in phase III interdigestive motility ofa human subject's intestinal tract. Increasing intestinal motility, forexample, by administration of a chemical prokinetic agent, preventsrelapse of the SIBO condition, which otherwise typically recurs withinabout two months, due to continuing intestinal dysmotility. Theprokinetic agent causes an in increase in phase III interdigestivemotility of the human subject's intestinal tract, thus preventing arecurrence of the bacterial overgrowth. Continued administration of aprokinetic agent to enhance a subject's phase III interdigestivemotility can extend for an indefinite period as needed to preventrelapse of the SIBO condition.

Preferably, the prokinetic agent is a known prokinetic peptide, such asmotilin, or functional analog thereof, such as a macrolide compound, forexample, erythromycin (50 mg/day to 2000 mg/day in divided doses orallyor I.V. in divided doses), or azithromycin (250-1000 mg/day orally).

However, a bile acid, or a bile salt derived therefrom, is anotherpreferred prokinetic agent for inducing or increasing phase IIIinterdigestive motility. (E. P. DiMagno, Regulation of interdigestivegastrointestinal motility and secretion, Digestion 58 Suppl. 1:53-55[1997]; V. B. Nieuwenhuijs et al., Disrupted bile flow affectsinterdigestive small bowel motility in rats, Surgery 122(3):600-08[1997]; P. M. Hellstrom et al., Role of bile in regulation of gutmotility, J. Intern. Med. 237(4):395-402 [1995]; V. Plourde et al.,Interdigestive intestinal motility in dogs with chronic exclusion ofbile from the digestive tract, Can. J. Physiol. Pharmacol.65(12):2493-96 [1987]). Useful bile acids include ursodeoxycholic acidand chenodeoxycholic acid; useful bile salts include sodium or potassiumsalts of ursodeoxycholate or chenodeoxycholate, or derivatives thereof.

A compound with cholinergic activity, such as cisapride, (i.e.,Propulsid®; 1 to 20 mg, one to four times per day orally or I.V.), isalso preferred as a prokinetic agent for inducing or increasing phaseIII interdigestive motility. Cisapride is particularly effective inalleviating or improving hyperalgesia related to SIBO or associated withdisorders caused by SIBO, such as IBS, fibromyalgia, or Crohn's disease.

A dopamine antagonist, such as metoclopramide (1-10 mg four to six timesper day orally or I.V.), domperidone (10 mg, one to four times per dayorally), or bethanechol (5 mg/day to 50 mg every 3-4 hours orally; 5-10mg four times daily subcutaneously), is another preferred prokineticagent for inducing or increasing phase III interdigestive motility.Dopamine antagonists, such as domperidone, are particularly effective inalleviating or improving hyperalgesia related to SIBO or associated withdisorders caused by SIBO, such as IBS, fibromyalgia, or Crohn's disease.

Also preferred is a nitric oxide altering agent, such as nitroglycerin,nomega-nitro-L-arginine methylester (L-NAME), N-monomethyl-L-arginine(L-NMMA), or a 5-hydroxytryptamine (HT or serotonin) receptorantagonist, such as ondansetron (2-4 mg up to every 4-8 hours I.V.;pediatric 0.1 mg/kg/day) or alosetron. The 5-HT receptor antagonists,such as ondansetron and alosetron, are particularly effective inimproving hyperalgesia related to SIBO, or associated with disorderscaused by SIBO, such as IBS, fibromyalgia, or Crohn's disease.

An antihistamine, such as promethazine (oral or I.V. 12.5 mg/day to 25mg every four hours orally or I.V.), meclizine (oral 50 mg/day to 100 mgfour times per day), or other antihistamines, except ranitidine(Zantac), famotidine, and nizatidine, are also preferred as prokineticagents for inducing or increasing phase III interdigestive motility.

Also preferred are neuroleptic agents, including prochlorperazine (2.5mg/day to 10 mg every three hours orally; 25 mg twice daily rectally; 5mg/day to 10 mg every three hours, not to exceed 240 mg/dayintramuscularly; 2.5 mg/day to 10 mg every four hours I.V.),chlorpromazine (0.25 mg/lb. up to every four hours [5-400 mg/day]orally; 0.5 mg/lb. up to every 6 hours rectally; intramuscular 0.25/lb.every six hours, not to exceed 75/mg/day), or haloperidol (oral 5-10mg/day orally; 0.5-10 mg/day I.V.). Also useful as a prokinetic agent,for purposes of the present invention, is a kappa agonist, such asfedotozine (1-30 mg/day), but not excluding other opiate agonists. Theopiate (opioid) agonists, such as fedotozine, are particularly effectivein alleviating or improving hyperalgesia related to SIBO or associatedwith disorders caused by SIBO, such as IBS, fibromyalgia, or Crohn'sdisease.

The preceding are merely illustrative of the suitable means by whichsmall intestinal bacterial overgrowth is at least partially eradicatedby treatment in accordance or in combination with the inventive methods.These means can be used separately, or in combination, by thepractitioner as suits the needs of an individual human subject.

Optionally, treating further includes administering to the human subjectan anti-inflammatory cytokine or an agonist thereof, substantiallysimultaneously with or after at least partially eradicating thebacterial overgrowth of the small intestine, to accelerate or furtherimprove the symptom(s) of irritable bowel syndrome, fibromyalgia,chronic fatigue syndrome, depression, ADHD, or an autoimmune disease, orCrohn's disease. Useful anti-inflammatory cytokines include human IL-4,IL-10, IL-11, or TGF-β, derived from a human source or a transgenicnon-human source expressing a human gene. The anti-inflammatory cytokineis preferably injected or infused intravenously or subcutaneously.

Optionally, when the suspected diagnosis is irritable bowel syndrome,fibromyalgia, chronic fatigue syndrome, depression, ADHD, man autoimmunedisease, such as multiple sclerosis or systemic lupus erythematosus,symptoms are improved by administering an antagonist of apro-inflammatory cytokine or an antibody that specifically binds apro-inflammatory cytokine. The antagonist or antibody is administered tothe human subject substantially simultaneously with or after treatmentto at least partially eradicate the bacterial overgrowth. The antagonistor antibody is one that binds to a pro-inflammatory cytokine orantagonizes the activity or receptor binding of a pro-inflammatorycytokine. Pro-inflammatory cytokines include TNF-α, IL-1α, IL-1β, IL-6,IL-8, IL-12, or LIF. The cytokine antagonist or antibody is preferablyderived from a human source or is a chimeric protein having a humanprotein constituent. The cytokine antagonist or antibody is preferablydelivered to the human subject by intravenous infusion.

Optionally, the method of treating irritable bowel syndrome,fibromyalgia, chronic fatigue syndrome, depression, attentiondeficit/hyperactivity disorder, an autoimmune disease, or Crohn'sdisease, further comprises administering an agent that modifies afferentneural feedback or sensory perception. This is particularly useful when,after at least partial eradication of SIBO, the subject residualsymptoms of hyperalgesia related to SIBO or associated with a disordercaused by SIBO, such as IBS, fibromyalgia, or Crohn's disease. Agentsthat modify afferent neural feedback or sensory perception include 5-HTreceptor antagonists, such as ondansetron and alosetron; opiateagonists, such as fedotozine; peppermint oil; cisapride; a dopamineantagonist, such as domperidone; an antidepressant agent; an anxiolyticagent; or a combination of any of these. Useful antidepressant agentsinclude tricyclic antidepressants, such as amitriptyline (Elavil);tetracyclic antidepressants, such as maprotiline; serotonin re-uptakeinhibitors, such as fluoxetine (Prozac) or sertraline (Zoloft);monoamine oxidase inhibitors, such as phenelzine; and miscellaneousantidepressants, such as trazodone, venlafaxine, mirtazapine,nefazodone, or bupropion (Wellbutrin). Typically, useful antidepressantagents are available in hydrochloride, sulfated, or other conjugatedforms, and all of these conjugated forms are included among the usefulantidepressant agents. Useful anxiolytic (anti-anxiety) agents includebenzodiazepine compounds, such as Librium, Atavin, Xanax, Valium,Tranxene, and Serax, or other anxiolytic agents such as Paxil.

Eradication of the bacterial overgrowth is determined by detectionmethods described above, particularly in comparison with recordedresults from pre-treatment detection. After at least partiallyeradicating the bacterial overgrowth, in accordance with the presentmethod, the symptom(s) of irritable bowel syndrome, fibromyalgia,chronic fatigue syndrome, depression, ADHD, an autoimmune disease, orCrohn's disease are improved. Improvement in a symptom(s) is typicallydetermined by self-reporting by the human subject, for example by VASscoring or other questionnaire. Improvement in academic, professional,or social functioning, e.g., in cases of ADHD or depression can also bereported by others or can be observed by the clinician. Improvement(increase) in pain threshold, e.g., in subjects diagnosed withfibromyalgia, can be measured digitally, for example, by tender pointcount, or mechanically, for example, by dolorimetry. (F. Wolfe et al.,Aspects of Fibromyalgia in the General Population: Sex, Pain Threshold,and Fibromyalgia Symptoms, J. Rheumatol. 22:151-56 [1995]). Improvementin visceral hypersensitivity or hyperalgesia can be measured by balloondistension of the gut, for example, by using an electronic barostat. (B.D. Nabiloff et al., Evidence for two distinct perceptual alterations inirritable bowel syndrome, Gut 41:505-12 {1997]). Some improvement(s) insymptoms, for example systemic lupus erythematosus symptoms, such asrashes, photosensitivity, oral ulcers, arthritis, serositis, orimprovements in the condition of blood, kidney or nervous system, can bedetermined by clinical observation and measurement.

The present invention also relates to a kit for the diagnosis of SIBO ora SIBO-caused condition. The kit comprises at least one breath samplingcontainer, a pre-measured amount of a substrate, and instructions for auser in detecting the presence or absence of SIBO by determining therelative amounts of methane, hydrogen, and at least onesulfur-containing gas in a gas mixture exhaled by the human subject,after the human subject has ingested a controlled quantity of thesubstrate. The present kit is useful for practicing the inventive methodof detecting SIBO in a human subject, as described hereinabove.

The kit is a ready assemblage of materials or components forfacilitating the detection of small intestinal bacterial overgrowth, inaccordance with the present invention. The kit includes suitable storagemeans for containing the other components of the kit. The kit includesat least one, and most preferably multiple, air-tight breath samplingcontainer(s), such as a bag, cylinder, or bottle, and at least onepre-measured amount of a substrate, which is preferably anisotope-labeled substrate or substrate that is poorly digestible by ahuman. Preferably the substrate is a sugar, such as lactulose (e.g.,10-20 g units) or xylose, or a sugar, such as glucose (e.g., 75-80 gunits), lactose, or sucrose, for measuring breath hydrogen, methane, andat least one sulfur-containing gas, such as hydrogen sulfide, asulfhydryl compound, methanethiol, dimethylsulfide, dimethyl disulfide,an allyl methyl sulfide, an allyl methyl sulfide, an allyl methyldisulfide, an allyl disulfide, an allyl mercaptan, or a methylmercaptan.

The present kit also contains instructions for a user in how to use thekit to detect small intestinal bacterial overgrowth (SIBO) or tocorroborate a suspected diagnosis of irritable bowel syndrome,fibromyalgia, chronic fatigue syndrome, chronic pelvic pain syndrome,autism, impaired mentation, impaired memory, depression, ADHD, anautoimmune disease, or Crohn's disease, in accordance with the presentmethods.

Optionally, the kit also contains compositions useful for at leastpartially eradicating SIBO, as described above.

The components assembled in the kits of the present invention areprovided to the practitioner stored in any convenient and suitable waythat preserves their operability and utility. For example the componentscan be in dissolved, dehydrated, or lyophilized form, they can beprovided at room, refrigerated or frozen temperatures.

The foregoing descriptions for the methods and kits of the presentinvention are illustrative and by no means exhaustive. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples.

EXAMPLES Example 1 Composition of the Database

Data were assembled from 202 human subjects from the Cedars-SinaiMedical Center GI Motility Program who completed an extensivequestionnaire of health history. These patients were all referred forlactulose breath hydrogen testing (LBHT) by more than 30 privategastroenterologists. These patients were selected by theirgastroenterologists to undergo breath testing, because they had symptomscompatible with SIBO. However, the questionnaire focused on general riskfactors, associated conditions, and symptoms found in these patients andnot specifically the incidence of SIBO. After antibiotic therapy, 59subjects actually returned for a follow up LBHT and a follow-upquestionnaire. This likely resulted in an underestimate ofresponsiveness to treatment, since only those who failed to respondadequately were likely to return to assess eradication of SIBO.

Example 2 Breath Hydrogen Testing

Subjects were tested after an overnight fast. At time zero, each subjectswallowed 15 ml of Chronulac formula, delivering 10 g of lactulose;every 5-20 min thereafter, for 2-4 hours, a 50 cm³ end-expiratory breathsample was taken with an airtight sampling bag. Each breath sample wasthen analyzed for hydrogen content with a gas chromatograph (QuintronModel DP, Quintron Instrument Co., Division of E.F. Brewer Co, MenomoneeFalls, Wis. 53051), standardized using a QuinGas standard as instructedby the manufacturer. Hydrogen peaks were plotted before and after anantimicrobial treatment regimen for comparison. The normal range for thesecond hydrogen peak was 0 to 20 ppm.

Example 3 Diagnosis and Antibiotic Treatment of Irritable Bowel Syndrome

The two hundred-two (202) human subjects were assessed for SIBO withLBHT. Of the 202 subjects in the database, 95 claimed to have been givena diagnosis of IBS. In addition, a symptom questionnaire was used todetermine whether these subjects fulfilled Rome criteria for IBS, andfour of the subjects failed to meet the Rome criteria. Crohn's diseasewas present in 14 of the subjects and four had a history of ulcerativecolitis. After these 22 subjects were excluded, 73 subjects remained.

Among the 107 subjects who stated that they had not previously beengiven a diagnosis of IBS, 78 met Rome criteria. After the 21 who hadCrohn's disease, five who had ulcerative colitis and one with shortbowel transit were excluded, 51 subjects remained. Data gathered fromthese subjects were pooled with data from the previous 73 subjects withsuspected IBS, yielding a total of 124 of the original 202 (61%)subjects with a suspected diagnosis of IBS.

Of the 124, 92 (74%) were positive for SIBO. However, of the 32 subjectsmeeting the Rome criteria, who were negative for SIBO, 14 had beentreated with antibiotics within 3 months prior to LBHT. Therefore, theincidence of SIBO among the 110 untreated subjects was 92 (84%), showinga strong association between a suspected diagnosis of IBS and thepresence of SIBO. After neomycin treatment (500 mg twice daily for tendays), 23 of these 92 returned for follow-up testing. On a visual analogscores (VAS), subjects were asked to rate their degree of post-treatmentimprovement. These 23 subjects reported a 60±31% improvement, although17 had only partial eradication of SIBO, based on their LBHT results.(FIG. 1).

There was a likely selection bias in the database due to the fact thatsubjects were referred for LBHT, because their physicians suspected theyhad SIBO. To correct for this bias, a pilot study was also conductedlooking at the incidence of bacterial overgrowth in patients with IBS.All patients between the ages of 18 and 65 referred to the Cedars-SinaiGI Motility Program who met Rome criteria for IBS, and who had had aprevious upper GI (small bowel) with follow-through (i.e., barium orGastrograffin imaging analysis) ruling out Crohn's disease andulcerative colitis, were asked to present to the GI motility laboratoryfor LBHT. Eight human subjects with a suspected diagnosis of IBS, basedon the Rome criteria, were tested for SIBO, using LBHT as described inExample 2. Seven of these patients (87.5%) were found to have SIBO basedon hydrogen peaks in a range of 80-250 ppm of hydrogen. Six of the 7subjects testing positive for SIBO returned approximately 10 days aftercompletion of a 10 day course of neomycin as described above. Neomycintreatment completely eradicated the SIBO in each of the six subjects,based on post-treatment breath hydrogen peaks in the normal range of0-20 ppm. The six subjects reported an average improvement in their IBSsymptoms of 65±28% (Range: 20-100%) on VAS scoring. FIG. 2 shows VAS forthe six subjects, based on a scale of 0-5, with 0 implying no pain and 5the most pain of life-time. It is clear from these results that at leastpartial eradication of bacterial overgrowth results in an improvement ingastrointestinal symptoms including bloating, gas, diarrhea, abdominalpain, sensation of incomplete evacuation and even constipation,associated with IBS. Additionally, significant extraintestinal symptomsof IBS, such as joint pain and fatigue, were also substantiallyimproved, and the degree of improvement was greater in subjects who hadcomplete eradication of SIBO.

Comparison of efficacies of various antibiotic regimes for treatingSIBO. Subjects referred to the Cedars-Sinai GI Motility Program for alactulose breath hydrogen test (LBHT) to assess SIBO were entered into adatabase. Those that tested positive for SIBO were given antibiotictreatment by their referring physician and in some cases, returned for afollow-up LBHT to assess eradication of SIBO. During the follow-up LBHT,subjects were asked which antibiotic they were given treat their SIBO.The eradication rate of each antibiotic was evaluated.

Of the 771 subjects in the database, 561 (73%) tested positive for SIBO.Of the 170 subjects who returned for a follow-up LBHT, 65 subjects wereexcluded because they did not specify or could not remember whichantibiotic they took. Based on the remaining 105 subjects, neomycin,augmentin, and ciprofloxacin were the most commonly prescribed, withneomycin being most successful. (See Table 1 below). Flagyl was arelatively poor choice by itself. None of the commonly used antibioticswas universally successful in eradicating overgrowth. Thus, Table 1shows that, while a number of antibiotics are able to eradicate SIBO,neomycin was most effective.

TABLE 1 Comparison of efficacies of various antibiotic regimes fortreating SIBO Number % Patients of Patients Total with SIBO SIBOEradicated Number Eradicated Neomycin 42 76 55 Flagyl 2 8 25Ciprofloxacin 3 6 50 Augmentin 2 4 50 Flagyl + Ciprofloxacin 1 2 —*Tetracycline 2 2 —* Doxycycline 1 1 —* Trovan 0 1 —* Neomycin/Biaxin +Amoxicillin 1 1 —* Neomycin + Ciprofloxacin 1 1 —* Tetracycline + Flagyl1 1 —* Neomycin + Flagyl 0 1 —* Biaxin 0 1 —* *Too few subjects todetermine percent success.Prevalence of SIBO in normal controls. The prevalence of SIBO in IBScompared to normal controls was determined as defined by the lactulosehydrogen breath test. Fifty-seven IBS subjects enrolled in a doubleblind placebo controlled trial and 9 normal controls underwent alactulose breath hydrogen test (LBHT) to diagnose SIBO. IBS subjects hadto meet Rome I criteria. Control subjects had to have none of the Rome Icriteria, based on telephone or in-person interviews. SIBO was definedas a greater than 20 ppm rise in H₂ concentration during the first 90minutes of lactulose breath hydrogen testing. The prevalence of SIBO inIBS subjects and controls was compared using Chi-square.

Of the 57 IBS subjects, 41 (72%) had SIBO. Of the 9 normal controls,only 1 subject (11%) had SIBO (χ²=9.9, OR=20.5, CI:2.2-481.8, p<0.01).These results confirm the association between IBS and SIBO as there is amuch higher prevalence of SIBO in IBS compared to normal controls.

Example 4 Diagnosis and Treatment of Fibromyalgia and Chronic FatigueSyndrome

Fibromyalgia: Of the 202 patients in the database, 37 (18%) had asuspected diagnosis of fibromyalgia. Of these 37, 28 tested positive forSIBO. However, of the nine who tested negative for SIBO, six had takenantibiotics within the preceding 3 months, and were excluded. Therefore,28 out of 30 (93%) of subjects with suspected fibromyalgia had SIBO,demonstrating a strong association between a suspected diagnosis offibromyalgia and the presence of SIBO.

After neomycin treatment (500 mg twice daily, 10-day course), ten ofthese 28 subjects returned, and post-treatment LBHT confirmed that SIBOhad been at least partially eradicated. These ten subjects reported a63±19% overall improvement in their symptoms by VAS scoring. FIG. 3compares the VAS scores for various symptoms reported by the subjectswith a suspected diagnosis of fibromyalgia before and after neomycintreatment. Symptoms included bloating, gas, diarrhea, joint pain andfatigue to treatment. Subjects were asked to identify the symptom mostimproved. Five subjects reported that pain was the most improved; threesubjects reported that the level of fatigue was most improved, and twoothers reported that their abdominal complaints improved the most. Therewas a negative correlation between the degree of improvement in the VASscoring and the amount of residual hydrogen peak seen in LBHT.(Pearson=−0.689, p=0.02; FIG. 4).

Subsequently, forty-six human subjects with FM (ACR criteria) entered adouble blind randomized placebo controlled trial. Each subject underwentLBHT, a tender point examination and completed a questionnaire at theinitial (baseline) and at every subsequent visit. Subjects wererandomized to receive neomycin (500 mg twice daily in liquid form) or amatched placebo, for 10 days. After completion of this treatment,subjects with persistent SIBO received antibiotics (open label) until atleast partially eradication was confirmed by LBHT. T-test was used tocompare the symptom scores of patients whose SIBO condition was at leastpartially eradicated with those whose SIBO was not at least partiallyeradicated.

Forty-two of the 46 FM patients (91.3%) were found to have SIBO. Six outof 20 patients (30%) in the neomycin group achieved complete at leastpartially eradication in the blinded arm. Only 6 subjects showed nodifference in the symptom score before and after the 10 d treatment.Twenty-eight subjects went on to open label treatment with 17 (60.7%)achieving complete at least partially eradication of SIBO. When symptomscores after at least partially eradication of SIBO on double blind oropen treatment were compared to baseline, there was significantimprovement in Tender Points, Tender Point Score, Hamilton DepressionScale, Fibromyalgia Impact Questionnaire (FIQ), Beck Depression Scale,Health Assessment Questionnaire (HAQ), VAS-Pain,VAS-Memory/Concentration and IBS-Quality of Life (QOL). (Initial data inTable 1a). These results confirm that SIBO is associated withfibromyalgia, and that at least partially eradication of SIBO improvessymptoms in fibromyalgia.

TABLE 1A Selected Symptom Scores Double Blind Randomized PlaceboControlled Trial with Subjects Diagnosed with Fibromyalgia. SIBO SIBOnot eradicated eradicated eradicated vs. not (n = 25) (p = 15)eradicated Observation Baseline eradicated P-value Baseline eradicatedP-value P-value Tender Points 13.3 ± 2.9 10.3 ± 4.2 0.01 13.6 ± 2.0 12.1± 4.1 NS NS (TP) TP Score 20.3 ± 7.0 15.0 ± 9.1 0.01 23.7 ± 8.0 19.9 ±9.7 NS NS FIQ 66.8 ± 18.2 49.5 ± 17.7 0.0001 72.7 ± 19.9 64.1 ± 20.90.04 0.02 VAS-pain(mm) 80.7 ± 22.7 52.4 ± 28.5 0.00005 87.5 ± 19.6 76.2± 25.2 NS 0.01 HAQ 42.4 ± 10.5 37.7 ± 10.1 0.005 45.1 ± 11.2 43.9 ± 12.1NS NSChronic Fatigue Syndrome: Thirty of 202 subjects in the database (15.9%)had received a diagnosis of chronic fatigue syndrome. Of these 30subjects, 21 (70%) had SIBO as indicated by LBHT, but four out of thenine without SIBO had recently taken antibiotics. Therefore, theprevalence of SIBO was 21 out of 26 (81%) subjects with a diagnosis ofCFS. After treatment with neomycin (500 mg twice daily, 10-day course),nine of the 21 subjects diagnosed with CFS, returned for follow-up LBHTand questionnaire. LBHT showed that all nine subjects experienced atleast partially eradication of SIBO, and important symptoms of CFS weresubstantially improved aftertreatment. (Table 2).

TABLE 2 VAS scores by CFS patients reporting before and afteranti-biotic treatment. Symptom Before Antibiotic After AntibioticP-value Bloating 4.3 ± 1.0 2.3 ± 1.7 0.002 Fatigue 4.6 ± 1.0 3.5 ± 1.40.02

Example 5 Autoimmune Diseases, Depression, ADHD, Autism, Mentation andMemory

SLE. Fifteen of the 202 (7.4%) subjects in the database had beendiagnosed with SLE. Of these 15 subjects, 13 (87%) had bacterialovergrowth, as indicated by LBHT. Four of the 15 subjects with SLEreturned for follow-up LBHT and questionnaire after treatment withneomycin (500 mg twice daily for 10 days). LBHT results for these fourwere negative for SIBO, and other significant symptoms weresignificantly improved after treatment. (Table 3).

TABLE 3 VAS scores by SLE patients reporting before and afteranti-biotic treatment. Symptom Before Antibiotic After AntibioticP-value Bloating 3.0 ± 2.0 1.3 ± 1.3 0.1 Joint Pains 2.5 ± 1.5 0.5 ± 0.60.04 Gas 3.3 ± 1.7 1.9 ± 1.7 0.3 Fatigue 4.6 ± 1.0 3.5 ± 1.4 0.3Multiple Sclerosis: A 22-year-old female who presented with a history ofmultiple sclerosis symptoms and with plaques demonstrated on MRIimaging. A suspected diagnosis of multiple sclerosis had been made by aneurologist was based on various neuropathies of the peripheral nervoussystem, including numbness, tingling, and weakness in the lowerextremities, but this subject also had associated bloating, gas,distension and alteration in bowel habits. The subject also complainedof a significant fatigue and nausea. The subject underwent LBHT, whichdetected SIBO. She was subsequently treated with neomycin (500 mg twicedaily for days), which at least partially eradicated the bacterialovergrowth. This was followed by complete resolution of her nausea,fatigue, bloating, gas distension and alteration in bowel habits. Inaddition, the subject showed dramatic improvement and resolution of herneuropathies. She no longer had numbness or tingling in the hands orfeet and was functioning quite well. Approximately 6-8 weeks after thisinitial response, the patient had a relapse of her symptoms, includingbloating, gas, distension and neuropathy. She had a repeat LBHT thatconfirmed a recurrence of SIBO. Upon re-treatment with neomycin (500 mgtwice daily for 10 days), she once again experienced complete resolutionof her symptoms.Depression: A 73-year-old female presented with bloating, gas, abdominaldistention, and cramping for a period of 3 years prior to LBHT. Symptomsof depression first appeared concurrently with the first appearance ofbowel symptoms, and were serious enough that psychiatric hospitalizationhad been considered by her attending psychiatrist. The subject reportedfeeling very depressed and was convinced that life was not worth living.The subject's LBHT indicated the presence of a SIBO condition. Aftertreatment with neomycin (500 mg twice daily for 10 days), the subjectstated that she felt “100% better.” She reported that her depression wascompletely resolved and that her energy was back to normal. In addition,her bowel symptoms were also completely improved. The subject had beenprescribed eight different anti-depressant medications, all of whichwere discontinued as a result of her improvement.ADHD: A 13-year-old female was brought in by her mother with a suspecteddiagnosis of attention deficit/hyperactivity disorder (AD type), made bya pediatrician. Concurrently, she also had significant bloating, gas andsome alteration in bowel habits. She had initially been referred fordiagnosis by her teachers and school counselors, because she had beenhaving difficulty performing in school for the previous two to threeyears, after having previously been a very good student. Prior to thedetection of SIBO, the subject had been treated with multiplepharmacologic agents for depression, including amitryptiline, with nonoticeable improvement in her symptoms.

The subject underwent LBHT that demonstrated the presence of SIBO. Thesubject was treated with neomycin (500 mg twice daily for 10 days) andafter complete at least partially eradication of the bacterialovergrowth, she had resolution of her bowel symptoms. Additionally, shestarted to get “A” averages in school again after being in the “C”range. She was able to concentrate better, and her teachers noticed adifference in her focus and attitude. Approximately two months later thesubject had a relapse in her attention problem which was concurrent witha recurrence of the bacterial overgrowth, as detected by LBHT. Afterrepeat treatment with neomycin (500 mg twice daily for 10 days), thesubject again responded with improved concentration and resolution ofbowel symptoms.

Autism: The patient was a 6-year-old female with a history of autismafter having failed development after the age of one year. Beforetreatment, the patient was categorized as having a developmental age of15 months. She also complained of abdominal distension, gas, bloatingand altered bowel habits. The patient was treated with Augmentin (500 mgtwice a day for ten days), which resulted in a substantial improvementin bowel habits altogether. The bloating, gas, distension and diarrhearesolved. In addition, there were some positive concentration andbehavioral changes. The patient was more responsive and cognitivelyappreciative of her parents' wishes, and there was some advancement inintellectual behavior. For example, after treatment she was able totolerate clothing and had improved concentration.Memory/Mentation/Concentration: The patient was a 72-year-old femalewith a history of chronic intestinal complaints over several years. Sheexperienced altered bowel habits with alternating diarrhea andconstipation with bloating, gas, distension and abdominal pain. Also,she had been diagnosed by several psychiatrists as having psychiatricproblems due to decreased mentation from mild senility, and shecontemplated psychiatric hospitalization.

SIBO was detected in this patient by LBHT. A subsequent course ofantibiotics completely eradicated the SIBO condition, and she returnedto report joyfully that she no longer needed the psychotropicmedications that she had been prescribed, because she feels completelynormal, including her bowels. She is now able to drive a car again,which was previously prevented from doing due to her impaired memory anddifficulty in concentrating on the road. Treatment of her SIBO condition(neomycin, 500 mg twice a day for ten days) has produced a dramaticimprovement in her quality of life.

Example 6 Diagnosis and Treatment of Crohn's Disease

Of the 202 subjects in the database, 39 (19%) had a suspected diagnosisof Crohn's disease. Of these 39, eight demonstrated short bowel transitand one subject produced neither hydrogen nor methane in LBHT; thesenine were excluded. Of the 30 remaining subjects, 22 had SIBO. However,of the eight subjects who had a negative LBHT result, five had beentreated with antibiotics within the preceding 3 months. If thesesubjects are excluded, 22 out of 25 (88%) subjects with a suspecteddiagnosis of Crohn's disease had SIBO, which shows a strong associationbetween a suspected diagnosis of Crohn's disease and the presence ofSIBO.

Of the 22 patients testing positive for the presence of SIBO, ninereturned after neomycin treatment (10-day course of 500 mg twice/daily)for LBHT, which showed at least partially eradication of SIBO. Thesenine patients reported a 57±32% (n=8 because one patient failed toreport percent improvement) overall improvement in their symptoms byVAS. If these subjects remained positive after antibiotic treatment withneomycin, metronidazole (Flagyl®), or ciprofloxacin, their improvementwas only 20±0% as opposed to 69±27% if the breath test was negative(p<0.05). FIG. 5 shows a dramatic improvement in the patients symptomsafter treatment. There was an especially notable reduction in bloodystools, diarrhea and fatigue.

As with the subjects with fibromyalgia, there was a negative correlationbetween the degree of improvement in the VAS scoring and the amount ofresidual hydrogen production (Pearson=−0.787, p=0.02; FIG. 6).

To correct for selection bias, a pilot study was conducted to determinethe incidence of SIBO in subjects who had received a suspected diagnosisof Crohn's disease at Cedars-Sinai Medical Center's IBD Center withinthe preceding three months. Six of these subjects underwent LBHT, ofwhom five (83%) were positive for SIBO.

Two of the six subjects returned for follow-up after antibiotic therapy(10-day course of neomycin). Post-treatment LBHTs showed that SIBO hadbeen completely at least partially eradicated in both subjects. Theyreported, respectively, a 60% and 80% overall improvement in theirsymptoms. This improvement was stated to include substantial reductionin diarrhea, gas and bloating.

Example 7 Response Stratification

There is a stratification in the degree of overgrowth and production ofhydrogen among the various diagnostic categories. For example, duringthe double blind study in the treatment of SIBO in fibromyalgia (Example4), it was noted that the level of hydrogen production during the LBHTwas much higher in this group of subjects as compared to those insubjects in the IBS incidence study described in Example 3. Given thatthe bacterial load is related to the level of hydrogen production, thisimplies that the degree of overgrowth is higher in patients withfibromyalgia compared to subjects with IBS.

The stratification of breath hydrogen levels with respect to diagnosticcategories is as follows: IBS/Crohn's Disease (40-70 ppm of hydrogen);CFS (50-100 ppm of hydrogen); and FM (100-250 ppm of hydrogen).

Example 8 Intestinal Dysmotility Associated with IBS and FM

Clinical experience showed that SIBO tends to recur after anti-biotictreatment within about 2 months. To demonstrate that a lack of phase IIIinterdigestive motility is responsible for SIBO in subjects with IBS orfibromyalgia, antreduodenal manometry was conducted in human subjectsdiagnosed with IBS or FM.

Antreduodenal Manometry. Phase III interdigestive (fasting) motility wasassessed in 15 human subjects. An antreduodenal manometry was performedby placing an 8-channel small bowel manometry catheter (each channelspaced 5 cm apart) into the small bowel using fluoroscopic guidance.After placement of the catheter, manometric recordings were made with anArndorffer perfusion system with signals collected usingMedtronics/Synectics Polygraf and associated Polygram software. Datawere assessed for the characteristics of interdigestive motility.IBS. Phase III interdigestive motility was assessed for a six-hourperiod in 15 human subjects having a suspected diagnosis of IBS, asdefined by Rome Criteria, corroborated by concomitant SIBO. Of these 15subjects, 13 (86%) had no detectable phase III interdigestive motilityduring the period of study. One subject (7%) had phase IIIinterdigestive motility of short duration (<3 minutes), and one subject(7%) had normal phase III interdigestive motility.Fibromyalgia. Phase III interdigestive motility was assessed in sevenhuman subjects having a suspected diagnosis of fibromyalgia corroboratedby the presence of SIBO. Of these seven subjects, six (86%) tackeddetectable phase III interdigestive motility, and one subject (14%) hadmotility of less than normal peristaltic amplitude. The duration ofstudy in the patients with fibromyalgia averaged 216±45 minutes in thefasting state.

Example 9a Treatment of SIBO-Related IBS with a Prokinetic Agent

Erythromycin, as a motilin agonist, can induce phase III ofinterdigestive motility. (E.g., M. J. Clark et al., Erythromycinderivatives ABT229 and GM 611 act on motilin receptors in the rabbitduodenum, Clin. Exp. Pharmacol. Physiol. 26(3):242-45 [1999]).Therefore, two subjects with recurrent IBS symptoms received prokinetictreatment with erythromycin.

The two subjects were a 55-year-old female and a 43-year-old female,both diagnosed with IBS. SIBO was detected in these subjects by LBHT.Antibiotic treatment of the SIBO resulted in greater than 90%improvement in symptoms. However, IBS symptoms recurred three to fourweeks later, concurrent with a return of the SIBO condition. Subsequentcourses of antibiotic treatment resulted in a similar pattern ofimprovement followed by a rapid recurrence of IBS symptoms in bothsubjects. Antreduodenal manometry was performed, demonstrating a lack ofphase III of interdigestive motility, and erythromycin (50 mg daily) wasprescribed to the subjects. The two subjects subsequently remained freeof IBS symptoms and SIBO for at least 18 months and six months,respectively.

These results demonstrate the effectiveness of prokinetic treatment witherythromycin in preventing the recurrence of SIBO and IBS symptoms insubjects diagnosed with IBS.

Example 9b Treatment of SIBO-Related IBS with a Supplemental PancreaticEnzyme

Supplementing food with pancreatic enzymes facilitates more efficientabsorption and digestion of food nutrients, thus allowing ingested foodto be absorbed higher up in the small intestine than otherwise. Thisleads to a relative deprivation of nutrients to the bacteria involved inthe SIBO condition. An example of this treatment modality occurred inthe case of a 19-year-old male who had longstanding history of alteredbowel habits, bloating, gas, distension and significant urge toevacuate. All of these symptoms were consistent with irritable bowelsyndrome (IBS). The patient was diagnosed as having SIBO based on theresults of LBHT. Subsequent to treatment with antibiotics, the patienthad significant improvement in his symptoms. However, his SIBO conditionbecame difficult to manage due to antibiotic resistance. An alternativetreatment regimen was prescribed, which involved the addition of apancreatic enzyme to the patient's food (10,000 Units human pancrease incapsules ingested immediately before each meal). With this therapy, thepatient reported that his gastrointestinal complaints have improved byapproximately 30-40%, corresponding to partial eradication of his SIBOcondition. Treatment was continued for at least eight months with acontinuation of the improvement in symptoms during that period.

Example 9c Excessive Methane Production in Subjects with SmallIntestinal Bacterial Overgrowth is Associated with Less Diarrhea

Bacterial metabolism is the major mechanism for the removal of hydrogenthat is produced during fermentation reactions of intestinal bacteria.Specifically, hydrogen is consumed in the production of methane and inthe reduction of sulfates to sulfides, with the 2 pathways beingmutually exclusive. Since intestinal sulfides are known to be damagingto intestinal epithelium, it was hypothesized that diarrhea may be aless prevalent symptom among patients with small intestinal bacterialovergrowth (SIBO) who test positive for methane (no damaging sulfidesproduced).

Subjects referred to the Cedars-Sinai GI Motility Program for LBHT wereentered into a database. Subjects were asked to rate symptoms ofbloating, diarrhea, constipation, abdominal pain, mucous in stool,incomplete evacuation, straining and urgency, on visual analogue scales(0-5, with 0 representing no symptoms). An ANOVA was used to comparesymptom scores between subjects producing no measured gases (onlysulfide producing bacteria), H₂ only, H₂ and CH₄, and CH₄ only, on theLBHT.

Of the 771 subjects in the database, 48 were excluded because theydemonstrated rapid transit on the LBHT. Of the 723 subjects remaining,514 were positive for SIBO and 43 were considered non-methane,non-hydrogen producers. Among the 514 who had SIBO, 435 (85%) producedH₂ only, 68 (13%) produced both H₂ and CH₄, and 11 (2%) produced CH₄only. The severity of diarrhea was highest in the non-H₂, non CH₄ and H₂only group with less in the H₂ and CH₄ group, and CH₄ only group. Therewas a significant difference between the three groups for diarrhea(p<0.00001 after Boneferroni correction). Urgency demonstrated the sametrend, but was not significantly different. All other symptoms were nodifferent. The severity of diarrhea symptoms is less in SIBO patientswho excrete methane (FIG. 7). In the non-methane-producers, greaterseverity of diarrheal symptoms likely reflects the reduction of sulfatesto sulfides as the alternate pathway for the remove of hydrogen.

Example 10 Treatment of SIBO-Related Hyperalgesia

An adult male subject with a suspected diagnosis of IBS was found tohave SIBO, as detected by LBHT. Anorectal manometry revealed rectalhypersensitivity in this subject. After eradication of his SIBOcondition with antibiotic treatment, a repeat anorectal manometry showedthat his rectal hyperalgesia had resolved.

Two adult female subjects with IBS required additional pharmacologicmanipulations to treat their SIBO-related hyperalgesia. In the firstcase, SIBO was eradicated by antibiotic treatment. However, the subjectcomplained of persistent feelings of rectal distension, consistent withresidual hyperalgesia related to SIBO. The subjected was thenadministered Colpermin (peppermint oil) capsules and Elavil (5 mg takenat night) that alleviated her SIBO-related hyperalgesic symptoms,presumably by reducing intestinal wall tension and decreasingmechanoreceptor activation.

The second female subject with a diagnosis of IBS was also found to haveSIBO, as detected by LBHT. Her SIBO was eradicated by a combinedtreatment with antibiotic, intestinal lavage with Go-Lytely, andcisapride (10 mg tid) to increase her abnormally low phase IIIinterdigestive motility. After eradication of SIBO, this subjectsimilarly complained of persistent SIBO-related hyperalgesic symptoms ofthe bowel. Administration of Colpermin (peppermint oil) thensuccessfully alleviated the hyperalgesia, presumably by reducing themechanoreceptor feedback for rectal distension.

Example 11 Treatment of SIBO Using Predigested Nutritional Formula

Based on the hypothesis that SIBO is promoted by nutritional componentsin food arriving at the distal gut, where they are used for carbon andenergy by bacterial populations responsible for the SIBO condition, tenpatients (8 female; 2 male; age range 17-64 years old; none having had abowel resection) each diagnosed with IBS in accordance with the RomeCriteria, and each having SIBO as determined by LBHT, were treated witha total enteral nutrition (TEN) formula, which is absorbed in theproximal gut (Vivonex® T.E.N.; Sandoz Nutrition, Minneapolis, Minn.).Vivonex is a glutamine-enriched total enteral nutrition product,containing protein as free amino acids in a 56:44 essential tononessential amino acid ratio, and inter alia, carbohydrate asmaltodextrin and modified starch, safflower oil, and all essentialvitamins and minerals. Vivonex is available as a powder for aqueousreconstitution (2.84 oz. packet; 1 packet mixed with 250 mL H₂O delivers300 mL of formula). Each patient was administered an amount ofreconstituted Vivonex to meet daily caloric needs according to themanufacturer's instructions, based on the each patient's weight, heightand other relevant factors. The patient's were allowed no othernutritional intake, but water was allowed freely. After 14 days of theTEN regimen, each patient resumed his or her normal diet.

FIG. 8 shows a representative result. In FIG. 8A (pre-treatment), SIBOwas initially detected by LBHT. After 14 days of the TEN regimen,follow-up LBHT shows that SIBO had been at least partially eradicated(FIG. 8B). Eradication was complete in eight of the patients with agreater than 80% improvement in IBS symptoms. Two of the patients hadonly partial eradication of SIBO with <20% improvement in IBS symptoms.The eradication of SIBO was maintained for up to two months after theTEN regimen was discontinued and normal nutrition had been resumed.

Example 12 Use of Active Lipids to Treat SIBO-Related Conditions

Oleate and Oleic Acid Slow Upper Gut Transit and Reduce DiarrheaPatients with Rapid Upper Gut Transit and Diarrhea

Rapid transit through the upper gut can result in diarrhea, maldigestionand absorption, and weight loss; and pharmacologic treatment withopiates or anticholinergics often is required. It was tested whetherfatty acids could be used to slow upper gut transit and reduce diarrheain patients with rapid transit and diarrhea.

In preliminary study, five patients with persistent diarrhea for 3 to 22months, (one each due to vagal denervation, ileal resection for Crohn'sdisease, and vagotomy and antrectomy, and two due to idiopathic causes)were studied. Each patient demonstrated rapid upper gut transit onroutine lactulose breath hydrogen testing (or variations thereofmeasuring labelled carbon dioxide) (Cammack et al. Gut 23:957-961[1982]). This test relies on the metabolism of certain carbohydratematerials (e.g. lactulose) by the microbial flora within the caecum. Bygenerating gas which can be detected in the expired air, it is possibleto make some estimation about the initial arrival of the administeredmaterial within the colon.

Each patient received orally in random order, 0, 1.6 or 3.2 g of sodiumoleate in 25 mL Ensure (Ross), followed by 100 mL water. Thirty minutesafter each dose of oleate, patients received 10 g lactulose orally,followed by 25 mL water. Breath samples were collected in commerciallyavailable breath testing bags (Quintron, Menomonee Falls, Wis.) every10-15 minutes, and the hydrogen content of the samples was measuredusing a breath analyzer (Microlyzer Model 12, Quintron Instruments,Menomonee Falls, Wis.), calibrated against gas samples of known hydrogenconcentration. With a syringe, a 40-mL sample of the expired breath waswithdrawn from the collection bag and analyzed immediately for hydrogenconcentration (ppm). The hydrogen concentration value from each samplewas plotted against time. Upper gut transit time was defined as the timein minutes from ingestion of lactulose (t₀) until a rise of H₂ of >10ppm. Data were further analyzed using 1-way repeated measures analysisof variance (ANOVA) (See Table 4).

TABLE 4 Effect of oleate on upper gut transit time (mean ± SE). Oleate(g) 0 1.6 3.2 Transit time (min) 46 ± 8.6 116 ± 11.1 140 ± 11.5

Upper gut transit was significantly prolonged by oleate in adose-dependent fashion (p<0.005, significant trend). During prolongedingestion of oleate 15-30 minutes prior to meals, all patients reportedreduced diarrhea. The patient with Crohn's disease reported completeresolution of chronic abdominal pain as well as post prandial bloatingand nausea, and gained 22 lbs. In addition, the patient with vagotomyand antrectomy reported resolution of postprandial dumping syndrome(flushing, nausea, light-headedness).

The effect of an active lipid on transit time was determined in 8 normalhuman subjects (1 male and 7 females with a mean age of 35.±.2.6 years[SE]) and 45 patients (20 males and 25 females with a mean age of49.1.±.2.5 [SE], age range from 18 to 90 years) with chronic diarrhea(i.e., continuous diarrhea for more than two months) associated with awide variety of diagnoses and conditions (e.g., Crohn's disease;irritable bowel syndrome; short bowel syndrome; Indiana pouch; AIDS;ulcerative colitis; vagotomy; antrectomy; ileostomy; partial andcomplete colectomy; colon cancer; diabetes mellitus type 1; pancreaticinsufficiency; radiation enteropathy; esophagectomy/gastric pull-up;total and subtotal gastrectomy; gastrojejunostomy), made by referringgastroenterologists. The method was the same as described above, exceptoleic acid (Penta Manufacturing, Livingston, N.J.) replaced sodiumoleate in 50 mL of Ensure emulsion. All subjects refrained from takingantibiotics for at least two weeks before each testing date and duringstool measurement periods. Patients were also instructed to refrain fromanti-diarrheal drugs, laxatives, somatostatin analogues oranticholinergics for at least 48 hours before each test. In both thenormal and patient groups, there was a significant slowing of upper guttransit time in response to oleic acid, as summarized in Table 5 below(p<0.001).

TABLE 5 Effect of Oleic Acid on upper gut transit time. Transit time(min) (mean ± SE) Oleic Acid (g) 0 1.6 3.2 Normal 105.2 ± 12.1   116 ±11.1   140 ± 11.5 Patients 29.3 ± 2.8 57.2 ± 4.5 83.3 ± 5.2

Continuing oleic acid treatment at home was offered to “responders”(i.e., patients who experienced a greater than 100% increase in baselinetransit time with 3.2 g oleic acid). Of the 36 responders of theoriginal 45 patients, 18 provided records of stool volume and frequencyon- and off-treatment for comparison. The inconvenient and unappealingnature of stool collection and measurement were the primary reasonsreported by responders who chose not to participate in stool collection.After completing a set of three preliminary breath hydrogen tests, eachparticipating responder was asked to refrain from taking oleic acid fortwo days in order to measure off-treatment stool output for a 24-hourperiod. Patients were issued a stool pattern record forth and a stoolcollection container with graduated volume markings to record thefrequency and volume of bowel movements. After two days without oleicacid, each patient took 3.2 g of oleic acid mixed with 25 mL of Ensureemulsion three times a day, 30 minutes before breakfast, lunch anddinner. After taking oleic acid for two days, patients recorded stooloutput for another 24-hour period. With this oleic acid emulsiontreatment, stool frequency decreased from 6.9±0.8 to 5.4±0.9 bowelmovements per 24-hour period (p<0.05), and stool volume decreased from1829.0±368.6 to 1322.5±256.9 per 24-hour period (p<0.05). A slight andtransient burning sensation in the mouth or throat was the only adverseeffect reported by any patient taking the oleic acid treatment.

These experiments demonstrate that active lipids, such as oleate andoleic acid, are effective in slowing upper gut transit in adose-dependent manner, thus enabling longer residence time for food inthe upper gut and a concomitant greater nutrient absorption there.

Fat in Distal Gut Inhibits Intestinal Transit More Potently than Fat inProximal Gut.

In 4 dogs equipped with duodenal (10 cm from pylorus) and mid-gut (160cm from pylorus) fistulas, as described hereinbelow (Example 14),intestinal transit was compared across an isolated 150 cm test segment(between fistulas) while 0, 15, 30 or 60 mM oleate was delivered intoeither the proximal or distal segment of the gut as a solution of mixedmicelles in pH 7.0 phosphate buffer at 2 mL/min for 90 minutes. Thesegment of gut not receiving oleate was perfused with phosphate buffer,pH 7.0, at 2 mL/min. 60 minutes after the start of the perfusion, ˜20μCi of ^(99m)Tc-DTPA (diethylenetriaminepentaacetic acid) was deliveredas a bolus into the test segment. Intestinal transit was then measuredby counting the radioactivity of 1 ml samples collected every 5 minutesfrom the diverted output of the mid-gut fistula.

Intestinal transit was calculated by determining the area under thecurve (AUC) of the cumulative percent recovery of the radioactivemarker. The square root values of the AUC (Sqrt AUC), where 0=norecovery by 30 minutes and 47.4=theoretical, instantaneous completerecovery by time 0, were compared across region of fat exposure andoleate dose using 2-way repeated measures ANOVA (see Table 6 below).

TABLE 6 Effect of Oleate and oleic acid on intestinal transit. Oleatedose (mM) Region (mean ± SE) of fat exposure 15 30 60 Proximal ½ of 41.6± 1.4 40.6 ± 10.2 34.4 ± 3.0 gut Distal ½ of gut 25.6 ± 1.4 18.9 ± 1.5  7.0 ± 3.8 Control: buffer into both proximal and distal ½ of gut = 41.4± 4.6.

These experiments demonstrate that intestinal transit is slower when fatis exposed in the distal ½ of gut (region effect p<0.01). Theseexperiments also demonstrate that oleate is effective to inhibitintestinal transit in a dose-dependent fashion (dose effect, p<0.05);and that dose dependent inhibition of intestinal transit by oleatedepends on the region of exposure (interaction between region and dose,p<0.01).

Case Study Showing Successful Treatment of Diarrhea-PredominantIrritable Bowel Syndrome With Oleic Acid. The patient was a 39-year oldmale with a history of adolescent-onset, persistent diarrhea. After aroutine gastrointestinal work-up failed to provide an explanation forhis symptoms, he was given the diagnosis of diarrhea-predominantirritable bowel syndrome. He presented with complaints of excessive gas,postprandial bloating, diarrhea and urgency, and 3 to 7 liquid bowelmovements per day. His upper gut transit times were (min) 30 (0 g oleicacid), 117 (1.6 g oleic acid) and 101 (3.2 g oleic acid). Withcontinuing oleic acid treatment as described above, he reported hisbowel frequency reduced to a single, solid bowel movement per day. Healso reported complete relief from the symptoms of gaseousness, bloatingand rectal urgency.Relatively rapid basal upper gut transit in Patients with InflammatoryBowel Disease (IBD). The mean upper gut transit time for IBD patients(n=18) at 0 grams of oleic acid was 79.1±11.0 min., compared to118.7±9.8 min for normal subjects (n=5) (p=0.04, t-test).Active lipid increases upper gut transit time. The mean transit time fornormal subjects (n=5) at 0 grams of oleic acid was 118.7±9.8 min, at 4grams of Oleic acid was 136.0±15.4 min. (P<0.05, t-test). The mean AUCfor normal subjects at 0 grams of oleic acid was 1438.9±208.5; at 4grams of oleic acid it was 1873.3±330.5 (p<0.05, t-test). The meantransit time for IBD patients (n=18) at 0 grams of oleic acid was79.1±11.0 min; at 4 grams of oleic acid it was 114.6±16.0 min. (p<0.05,t-test). The mean AUC for IBD patients at 0 grams of oleic acid was687.3±98.2; at 4 grams of oleic acid it was 1244.9±250.4. (p<0.05,t-test).

These data show that oleic acid slowed gut transit time and thussubstantially increased the opportunity for absorption of food nutrientsin the upper gut region in both normal and IBD groups. Thus, the inindividuals having SIBO a condition, treatment in accordance with themethod of deprives the bacteria of much of the nutrient simply requiredfor growth.

Example 13 Eradication of SIBO in Subjects with Irritable Bowel SyndromeLowers Their Serum Levels of 5-HT

Previous studies have shown that patients with irritable bowel syndrome(IBS) have elevated plasma 5-hydroxytryptamine (5-HT) levels. Since itwas shown hereinabove that IBS is associated with small intestinalbacterial overgrowth (SIBO) and symptoms of IBS are reduced byantibiotic eradication of SIBO, the hypothesis was tested thateradication of SIBO will reduce plasma 5-HT levels in IBS patients toprovide further evidence of the relationship between IBS and SIBO.

The plasma 5-HT levels of 7 human subjects diagnosed with IBS werecompared before and after successful eradication of SIBO, as part of adouble blind placebo controlled trial. A lactulose breath hydrogen test(LBHT) was performed to diagnose SIBO at baseline and when eradicationwas achieved. Fasting blood samples were taken at baseline and on theday that eradication of SIBO was confirmed. The plasma 5-HT level(ng/mL) was determined in each sample by ELISA (Kit-Research DiagnosticsInc., Flanders, N.J.). A paired t-test was performed to compare 5HTlevels (mean±SE) before and after eradication of SIBO.

The results indicated that the amount of plasma 5-HT was reduced from0.7±0.4 ng/mL before eradication to 0.5±0.5 ng/mL after eradication ofSIBO in the subjects (p<0.05). Thus, eradication of SIBO in IBS subjectsdecreases fasting plasma 5-HT levels, which provides further evidencefor the relationship between IBS and SIBO.

Example 14 Neural Regulation of the Rate of Upper GastrointestinalTransit

The experiments described below are based on a previously describedchronic multi-fistulated dog model, employing surgically fistulated maleor female mongrel dogs weighing about 25 kg each. (Lin, H. C. et al.,Inhibition of gastric emptying by glucose depends on length of intestineexposed to nutrient, Am. J. Physiol. 256:G404-G411 [1989]). The smallintestines of the dogs were each about 300 cm long from the pylorus tothe ileal-cecal valve. The duodenal fistula was situated 15 cm from thepylorus; the mid-gut fistula was situated 160 cm from the pylorus.Occluding Foley catheters (balloon catheters that are inflated toproduce a water-tight seal with the lumenal surface) were placed intothe distal limb of a duodenal fistula and a mid-gut fistula, fat orother test agents were administered lumenally to the thuscompartmentalized “proximal” section of the gut, i.e., between thefistulas, or to the compartmentalized “distal” section of the gut, i.e.,beyond the mid-gut fistula. Perfusate was pumped into a test sectionthrough the catheter at a rate of 2 mL/minute. Test agents wereadministered along with buffer perfusate, but some test agents wereadministered intravenously, where specifically noted.

Intestinal transit measurements were made by tracking the movement of aliquid marker across the approximately 150 cm intestinal test segment bydelivering about 20 μCi ^(99m)Tc chelated to diethyltriamine pentaaceticacid (DTPA) (Cunningham, K. M. et al., Use of technicium-99m (V)thiocyanate to measure gastric emptying of fat, J. Nucl. Med. 32:878-881[1991]) as a bolus into the test segment after 60 minutes of a 90-minuteperfusion. The output from the mid-gut fistula was collected every 5 minthereafter for 30 minutes, which period is illustrated in FIGS. 9-23.Using a matched dose of ^(99m)Tc to represent the original radioactivity(Johansson, C., Studies of gastrointestinal interactions, Scand. J.Gastroenterol. 9(Suppl 28):1-60 [1974]; Zierler, K., A simplifiedexplanation of the theory of indicator dilution for measurement of fluidflow and volume and other distributive phenomena, Bull. John Hopkins103:199-217 [1958]), the radioactivity delivered into the animal as wellas the radioactivity of the recovered fistula output were all measuredusing a gamma well counter. After correcting all counts to time zero,intestinal transit was calculated as the cumulative percent recovery ofthe delivered ^(99m)Tc-DTPA. This method has been well validated overthe years and appreciated for its advantage of minimal inadvertentmarker loss. To demonstrate this point, we perfused phosphate buffer, pH7.0, through the proximal gut and followed the cumulative recovery ofthis marker (% recovery) over time (n=1). There was a very high level ofmarker recovery, with 90% of the marker recovered by 30 minutes and 98%of the marker recovered by 45 minutes.

(1) Slowing of intestinal transit by PYY depends onondansetron-sensitive 5-HT-mediated pathway. Peptide YY (PYY) slowstransit and is a signal for lumenal fat (Lin, H. C. et al., Fat-inducedileal brake in the dog depends on peptide YY, Gastroenterol.110(5):1491-95 [1996b]; Lin, H. C. et al., Slowing of intestinal transitby fat in proximal gut depends on peptide YY, Neurogastroenterol.Motility 10:82 [1998]). Since serotonin (5-HT) can also be a signal forfat (Brown, N. J. et al., The effect of a 5HT3 antagonist on the ilealbrake mechanism in the rat, J. Pharmacol. 43:517-19 [1991]; Brown, N. J.et al. [1993]), the hypothesis was tested that the slowing of transit byPYY can depend on a 5-HT-mediated pathway by comparing the rate ofmarker transit during the administration of PYY in the presence orabsence of ondansetron (Ond; a 5-HT receptor antagonist) in the proximalversus distal gut (n=2 for each treatment).

Normal saline (0.15 M NaCl) or PYY (0.8 μg/kg/h) was administeredintravenously over a 90 minute period, while phosphate buffer, pH 7.0,was perfused into the lumen of the proximal gut through the duodenalfistula at a rate of 2 mL/min for the 90 minutes and was recovered fromthe output of the mid-gut fistula. The results are summarized in FIG. 9.Transit was slowed by intravenous PYY, with recovery of the markerdecreased from 75.1±3.6% (control: IV normal saline [NS]+lumenal normalsaline, i.e., NS-NS in FIG. 9) to 17.1±11.0% (IV PYY+lumenal normalsaline, i.e., PYY-NS in FIG. 9). This effect was abolished by adding thespecific 5-HT3 receptor antagonist ondansetron (0.7 mg/kg/h) to thebuffer introduced into the proximal gut so that recovery increased to78.3±4.8% (IV PYY+lumenal Ond proximal, i.e., PYY-Ond in prox in FIG. 9)but not by ondansetron in the distal gut, which decreased recovery to12.9±12.9% (IV PYY+Ond in Distal, i.e., PYY-Ond in Dist). These resultsimply that slowing of transit by PYY depended on a 5-HT-mediated pathwaylocated in the segment of the small intestine where transit wasmeasured.

(2) The fat induced jejunal brake depends on an ondansetron-sensitiveserotonin (5-HT)-mediated pathway. The hypothesis was tested thatslowing of transit by fat depends on a serotonergic pathway by comparingintestinal transit during perfusion with buffer or oleate in thepresence or absence of ondansetron, a 5-HT3 receptor antagonist, in theproximal gut (n=3 each treatment). Buffer or 60 mM oleate was perfusedthrough the duodenal fistula into the lumen of the proximal gut for a90-minute period, in the manner described in Example 14(1), along with abolus of normal saline±ondansetron (0.7 mg/kg) at the start of transitmeasurement. The rate of intestinal transit was slowed by the presenceof oleate (p<0.05) in an ondansetron-sensitive manner. (p<0.05). Theresults are summarized in FIG. 10.

Specifically, ondansetron increased recovery of marker in the perfusatefrom 41.6±4.6% (mean±SE) (lumenal oleate+lumenal normal saline, i.e.,Oleate-NS in FIG. 10) to 73.7±10.6% (lumenal oleate+lumenal ondansetron,i.e., Oleate-Ond in FIG. 10) during oleate perfusion but decreasedrecovery from 96.0±4.0% (lumenal phosphate buffer+lumenal normal saline,i.e., Buffer-NS in FIG. 10) to 57.9±15.9% (lumenal buffer+lumenalondansetron, i.e., Buffer-Ond in FIG. 10) during buffer perfusion. Theseresults imply that slowing of intestinal transit by the fat-inducedjejunal brake and the acceleration of intestinal transit by bufferdistension both depended on an ondansetron-sensitive 5-HT3-mediatedpathway.

(3) The fat-induced ileal brake depends on an ondansetron-sensitive,efferent serotonin (5-HT)-mediated pathway. The fistulated dog modelallows for the ileal brake (oleate in distal gut, buffer in proximalgut) to be separated into the afferent (distal) vs. efferent (proximal)limb of the response. Since 5-HT3 receptors are found on extrinsicprimary sensory neurons (afferent limb) and on intrinsic 5-HT neurons ofthe myenteric plexus (5-HT interneuron) (efferent limb), theidentification of the location of the 5-HT3 pathway (afferent vsefferent limb) can localize the serotonergic pathway responsible for theslowing of transit by fat in the distal gut (ileal brake). Usingoccluding Foley catheters, the small intestine was compartmentalizedinto the proximal gut and the distal gut as described hereinabove.Intestinal transit was measured across the proximal gut (betweenfistulas) as described hereinabove. By perfusing buffer through theproximal gut while fat was perfused through the distal gut to triggerthe fat-induced ileal brake, the distal gut represented the afferentlimb of the response and the proximal gut represented the efferent limbof the response. To test for the location of the serotonergic pathway,5-HT3 receptor antagonist ondansetron was then mixed with theappropriate perfusate and administered into either the proximal ordistal gut. Control=buffer in proximal and distal gut. Four dogs weretested.

Delivering ondansetron lumenally into either the proximal or distal gut,intestinal transit was slowed by the ileal brake (76.3±3.1% [Control inFIG. 11] vs. 22.9±3.8% [Ileal Brake in FIG. 11]; p<0.005). But the ilealbrake was abolished by ondansetron delivered to the proximal gut(68.5±2.7%; Ond in Prox in FIG. 11; n=4) but not distal gut (22.8±2.6%;Ond in Dist in FIG. 11; n=4).

Since ondansetron delivered with the fat in the distal gut had noeffect, but ondansetron delivered with the buffer in the proximal gutabolished the ileal brake, the slowing of intestinal transit by fat inthe distal gut depended on an ondansetron-sensitive, serotonergicpathway located on the efferent rather than afferent limb of theresponse. And since ondansetron abolished the jejunal brake in Example14(2) when delivered with fat and abolished the ileal brake in Example14(3) when delivered with buffer, this region-specific result cannot beexplained by inactivation of drug by fat, differences in permeability orabsorption.

(4) Ondansetron abolishes the fat-induced ileal brake in adose-dependent manner. The fat-induced ileal brake was abolished by the5-HT receptor antagonist ondansetron in a dose-dependent manner.Perfusion of buffer was through both the duodenal and mid-gut fistulas(2 mL/min over 90 minutes); the buffer administered to the mid-gutfistula contained buffered normal saline (pH=7.0; Buffer Control in FIG.12) or 60 mM oleate to induce the ileal brake response (Ileal Brake inFIG. 12). During the ileal brake response, ondansetron was added at t₀as a single bolus in the following doses (mg): 6.25; 12.5; and 25.Results are shown in FIG. 12.

Oleate induced the ileal brake (24.1% marker recovery [Ileal brake inFIG. 12] vs. 81.2% marker recovery for the Buffer Control). The ilealbrake was abolished by ondansetron delivered into the proximal gut in adose-dependent manner (35.4% marker recovery at 6.25 mg ondansetron,55.8% marker recovery at 12.5 mg ondansetron, and 77.6% marker recoveryat 25 mg ondansetron).

(5) Fat in the distal gut causes the release of 5-HT from the proximalgut. To test the hypothesis that fat in the distal gut causes therelease of 5-HT in the proximal gut, the amount of 5-HT collected fromthe output of the mid-gut fistula (proximal gut 5-HT) over a 90-minuteperiod of buffer perfusion through both the duodenal and mid-gutfistulas (2 mL/min); buffer (control) or oleate (60 mM) was administeredto the distal gut (n=1). The amount of 5-HT was determined using anELISA kit specific for 5-HT (Sigma; Graham-Smith, D. G., The carcinoidsyndrome, In: Topics in Gastroenterology, Truclove, S. C. and Lee, E.(eds.), Blackwells, London, p. 275 [1977]; Singh, S. M. et al.,Concentrations of serotonin in plasma—a test for appendicitis?, Clin.Chem. 34:2572-2574 [1988]). The amount of 5-HT released by the proximalgut increased in response to fat in the distal gut from 100 μg in thecontrol (buffer minus oleate) to 338 μg (buffer plus oleate to distalgut), showing that 5-HT is released in the proximal gut in response tofat in the distal gut. Thus, the release of 5-HT by the proximal gut canserve as a relayed signal for fat in the distal gut. The relayed releaseof 5-HT in the proximal gut in response to fat in the distal gut isconsistent with Example 14(2), showing that slowing of intestinaltransit by fat depends on an efferent 5-HT-mediated pathway to theproximal gut.(6) Ondansetron abolishes the fat-induced ileal brake when administeredlumenally but not intravenously. To confirm that the reversal of theslowing of transit by ondansetron was peripheral, i.e., enteric, ratherthan systemic, the effect of ondansetron was compared when deliveredluminally (through the duodenal fistula into the proximal gut) versusintravenously. Ondansetron was either delivered lumenally into theproximal gut (0.7 mg/kg/h; Ond in prox in FIG. 13) or administeredintravenously (0.15 mg/kg/1.5 h; iv Ond in FIG. 13) during fat-inducedileal brake (60 mM oleate input through the mid-gut fistula into thedistal gut as described above). Two dogs were tested (n=2).

Results are shown in FIG. 13. Compared to the ileal brake (20±1.8%marker recovery), the marker recovery increased to 78±2.4% with lumenalondansetron (p<0.005). Intravenous ondansetron had no substantial effecton the ileal brake (13±2.0% marker recovery). These results imply thatthe 5-HT3 receptor antagonist worked enterically rather thansystemically.

(7) The slowing of intestinal transit by distal gut 5-HT depends on anondansetron-sensitive 5-HT3-mediated pathway in the proximal gut(efferent) and distal gut (afferent).

To test the hypothesis that lumenal 5-HT may slow intestinal transit via5-HT3 receptors similar to fat, 0.7 mg/kg ondansetron, a 5-HT3 receptorantagonist or buffered saline (pH 7.0) was delivered into either theproximal or distal gut as a bolus at the start of the transitmeasurement. Four dogs were tested.

Results are shown in FIG. 14. The slowing of intestinal transit by 5-HT(0.1 mg/kg/h) administered to the distal gut (35.2±2.2% marker recovery)(vs. 76.1±4.7% marker recovery for buffer control) was abolished byondansetron added to the proximal or distal gut as shown by % markerrecovery of 73.8±9.5% (Ond-Prox in FIG. 14) vs. 79.5±2.4% (Ond-Dist inFIG. 14), respectively (p<0.001).

This shows that in the conscious whole animal, the slowing of intestinaltransit by luminal 5-HT depended on an ondansetron-sensitiveserotonergic pathway located on both the afferent and efferent limb ofthe intestine-intestinal reflex. (See also, Brown, N. J. et al.,Granisetron and ondansetron: effects on the ileal brake mechanism in therat, J. Pharm. Pharmacol. 45(6):521-24 [1993]). In contrast, the slowingof intestinal transit by distal gut fat (Example 14[3]) depended on a5-HT3 pathway localized specifically on the efferent limb to suggestthat 5-HT is not the stimulus for the afferent limb of the fat inducedileal brake, but rather involves a signal other than 5-HT, such as PYY.However, 5-HT is the stimulus for the afferent limb of the slowing ofintestinal transit by 5-HT in the distal gut.

(8a) 5-HT in the distal gut slows intestinal transit in a dose-dependentmanner. In a preliminary experiment, intestinal transit during bufferperfusion of both the proximal and distal guts (81.2% recovery) wasslowed by 5-HT in distal gut to that marker recovery decreased to 73.8%at 2 mg 5-HT (0.033 mg 5-HT/kg/h), 53.1% at 3 mg (0.05 mg 5-HT/kg/h) and11.6% at 4 mg (0.066 mg 5-HT/kg/h) dose over a 90 minute period (n=1).

The dose-dependent effect of 5-HT in slowing intestinal transit wasconfirmed in an additional experiment. The cumulative % recovery of theradioactive marker was reduced in a dose-dependent fashion as the 5-HTperfusion increased from 0 to 0.1 mg/kg/h to suggest that intestinaltransit is slowed by lumenal 5-HT. However, the speed of transit wasmarkedly accelerated when the 5-HT dose was increased to 0.3 mg/kg/h.(Table 7).

TABLE 7 Effect of 5-HT delivered to distal gut on intestinal transittime (min) in multi-fistulated dogs (n = 2 dogs). 5-HT dose (mg/kg/h ×90 min) 0 0.033 0.05 0.066 0.1 0.3 68.5 ± 1.0 69.6 ± 4.2 33.5 ± 1.5 15.2± 0.5 16.1 ± 4.9 73.8 ± 0.6(8b) Lumenal 5-HT, delivered to the proximal gut, slows intestinaltransit in a dose-dependent fashion in the conscious whole animal model.In in-vitro models, lumenal 5-HT applied to an isolated bowel loopaccelerated transit by triggering the peristaltic reflex. In contrast,in the conscious whole animal model applied herein (with extrinsicnerves intact), 5-HT applied lumenally slowed intestinal transit(Example 14[8a] above). In further experiments, 5-HT was delivered at arate of 0, 0.033, 0.066, 0.05 and 0.1 mg/kg/h into the proximal gut.Four dogs were tested.

Results are shown in FIG. 15. Intestinal transit was significantlyslowed by 5-HT in the proximal gut in a dose-dependent fashion(p<0.00001). Marker recovery during buffer perfusion was 75.0±4.4% whileat the dose of 0.066 mg/kg/h marker recovery was reduced to 16.9±3.7%,and was not significantly different from the dose of 0.1 mg/kg/h. At theintermediate dose of 0.05 mg/kg/h, marker recovery was 33.2±14.0%;buffer vs 0.05 mg/kg/h; p<0.005) and at the lowest dose of 0.033mg/kg/h, marker recovery was not significantly different from the buffercontrol.

(8c) Slowing of Intestinal Transit by 5-HT is not dependent on volume ofthe output of the midgut fistula. 5-HT stimulates small bowel andcolonic secretion. We have observed a slowing effect of 5-HT onintestinal transit (Example 14[8a-b]). As a control, to determinewhether intestinal transit correlates with volume of the output of themidgut fistula. Varying doses of 5-HT (0, 0.033, 0.1, 0.3 mg/kg/h) wereperfused into the proximal gut, ^(99m)Tc was delivered into the testsegment as a bolus for transit measurement. The volume of the output ofthe midgut fistula was collected during the last 30 minutes of the 90min perfusion experiment (n=21). Transit was plotted against outputvolume. There was no correlation between transit during 5-HT treatmentand the volume of the output of the midgut fistula (data not shown).

Therefore, the observed transit effect of 5-HT cannot be explainedsolely on the basis of volume effect related to 5-HT induced intestinalsecretion. The observed transit effect of 5-HT must depend ontransit-specific regulation.

Together, the results in Example 14(8) and show that, contrary to theeffect of 5-HT in an in-vitro model, lumenally administered 5-HT slowsintestinal transit in a dose-dependent fashion in the conscious wholeanimal model, which implies that the slowing of intestinal transitdepends on extrinsic nerves.

(9a) 5-HT in the distal gut causes release of 5-HT in the proximal gut.To test the hypothesis that 5-HT in the distal gut causes the release of5-HT in the proximal gut, the amount of 5-HT collected from the outputat the mid-gut fistula (Proximal gut 5-HT) over a 90-minute period ofbuffer perfusion through both the duodenal and mid-gut fistulas (2mL/min each) was compared in the presence or absence of 5-HT (0.05mg/kg/h) administered to the distal gut (n=1). 5-HT concentration wasdetermined using an ELISA kit specific for 5-HT (Sigma). The amount of5-HT released by the proximal gut increased from 156 μg in the control(minus distal 5-HT) to 450 μg (plus 5-HT to distal gut), implying that5-HT is released by the proximal gut in response to 5-HT in the distalgut. Thus, the release of 5-HT by the proximal gut can serve as arelayed signal for distal gut 5-HT. This relayed release of 5-HT in theproximal gut explains the results of Example 14(6) showing that theslowing of intestinal transit by distal gut 5-HT was abolished byondansetron in the proximal gut (efferent limb of response) as well asin the distal gut (afferent limb of response).(9b) Fat in distal gut releases 5-HT from proximal gut. To test thehypothesis that the proximal gut releases 5-HT in response to lipid inthe distal gut, we compared the amount of 5-HT in the output of themidgut fistula (i.e., proximal gut 5-HT) with buffered saline (control)or oleate in the distal gut. The amount of 5-HT collected over 90 minwas measured using a 5-HT-specific ELISA test kit, as described hereinabove. Four dogs were tested.

The amount of proximal gut 5-HT increased from 82.7±20.53 ng to211.75±35.44 ng (p<0.005) when the distal gut perfusate was switchedfrom buffer to oleate, implying that 5-HT is released from the proximalgut in response to fat in the distal gut, as a relayed signal for fat.

Fat is also a chemical trigger for the release of 5-HT, thus theseresults are consistent with the release of 5-HT via a long distance,intestino-intestinal communications, or reflex.

(9c) Luminal 5-HT slows intestinal transit via activation of theintestino-intestinal reflex. To confirm that 5-HT, delivered lumenally,slowed intestinal transit via the activation of an intestino-intestinalreflex, we compared intestinal transit across the proximal one-half ofgut while 0 (pH 7.0 buffered saline control) or 0.1 mg/kg/h of 5-HT wasdelivered into either the proximal or distal one-half of the gut. Fourdogs were tested.

Results are shown in FIG. 16. Intestinal transit across the proximal gutwas slowed by 5-HT in either the proximal or distal gut, demonstrated bythe marker recovery decreasing from 85.0±7.3% (Saline-Prox in FIG. 16)(p<0.005) to 20.1±4.5% for proximal gut 5-HT (5-HT-Prox in FIG. 16) and76.1±1.3% (Saline-Dist in FIG. 16) to 35.2±2.3% (5-HT-Dist in FIG. 16)(p<0.005) for distal gut 5-HT.

These results imply that the slowing of intestinal transit by 5-HTdepends on a long-distance, region-to-region reflex, since 5-HTadministered into the distal gut slowed intestinal transit through thephysically separate proximal gut.

(10) Intravenous PYY causes release of 5-HT in the proximal gut. Theamount of 5-HT released from the proximal gut in response to intravenousPYY or buffered saline (Control) during buffer perfusion (2 mL/min over90 minutes) through both the duodenal and mid-gut fistulas was measuredto test the hypothesis that intravenous PYY (0.8 mg/kg/h) causes therelease of 5-HT in the proximal gut. 5-HT was measured as in Example14(9) above. The amount of 5-HT released by the proximal gut increasedfrom 140.1 μg (Control) to 463.1 μg in response to intravenous PYY.

This result was comparable with the response when 60 mM oleate wasadministered to the distal gut (buffer only to the proximal gut) duringthe perfusion without intravenous PYY (509.8 μg of 5-HT; n=1), whichimplies that the release of 5-HT in the proximal gut stimulated by fatin the distal gut can be mediated by PYY.

(11) Slowing of intestinal transit by fat in the distal gut depends onan extrinsic adrenergic neural pathway. A distension-inducedintestino-intestinal inhibitory neural reflex projects through theceliac prevertebral celiac ganglion via a cholinergic afferent and anadrenergic efferent (Szurszewski, J. H. and King, B. H., Physiology ofprevertebral ganglia in mammals with special reference to interiormesenteric ganglion, In: Handbook of Physiology: The GastrointestinalSystem, Schultz, S. G. et al. (eds.), American Physiological Society,distributed by Oxford University Press, pp. 519-592 [1989]). Intestinaltransit was measured during fat perfusion of the distal small intestinein the presence or absence of intravenous propranolol (50 μg/kg/h; n=2dogs), a β-adrenoceptor antagonist, to test the hypothesis that theslowing of intestinal transit by fat in the distal gut also depends onan adrenergic pathway. Perfusion of buffer was through both the duodenaland mid-gut fistulas (2 mL/min over 90 minutes); the buffer administeredto the mid-gut fistula contained 60 mM oleate. The results areillustrated in FIG. 17.

Intestinal transit was slowed by distal gut fat (79.7±5.8% markerrecovery [Buffer Control in FIG. 17] compared to 25.8±5.2% recovery withfat perfusion into the distal gut [Oleate-NS in FIG. 17]). Intravenouspropranolol abolished this jejunal brake effect so that recoveryincreased to 72.1±4.7% (oleate+propanolol, i.e., Oleate-Prop in FIG.17), implying that the slowing of transit by fat in the distal gutdepends on a propranolol-sensitive, adrenergic pathway. This resultsupports the hypothesis that the response to fat involves an adrenergicefferent, such as the extrinsic nerves projecting through theprevertebral ganglia.

(12) Slowing of intestinal transit by PYY depends on an extrinsicadrenergic neural pathway. Intestinal transit during buffer perfusion ofthe proximal and distal small intestine in the presence or absence ofintravenous propranolol (50 μg/kg/h; n=2) was measured, to test thehypothesis that the slowing of intestinal transit by PYY (a fat signal)also depends on an adrenergic pathway. Perfusion was through bothfistulas as described in Example 14(11) except that oleate was notadministered to the distal gut, and, instead, 30 μg PYY (0.8 mg/kg/h)was administered intravenously during the 90 minute perfusion period.The results are summarized in FIG. 18.

Slowing of intestinal transit by PYY (78.1±2.2% marker recovery minusPYY [Buffer Control in FIG. 18] vs. 11.8±5.4% recovery with intravenousPYY [PYY-NS in FIG. 18]) was abolished by intravenous propranolol. Inthe presence of propanolol, marker recovery increased to 66.3±3.1%(PYY-Prop in FIG. 18). This result, consistent with the results ofExample 14(11), implies that the slowing of transit by PYY depends on apropranolol-sensitive, adrenergic pathway, which supports the hypothesisthat the response to PYY involves an adrenergic efferent such as theextrinsic nerves projecting through the prevertebral ganglia.

(13) Slowing of intestinal transit by 5-HT in the distal gut depends ona propranolol-sensitive extrinsic adrenergic neural pathway. Intestinaltransit during buffer perfusion of the proximal and distal smallintestine in the presence or absence of intravenous propranolol (50μg/kg/h; n=2) was measured, to test the hypothesis that the slowing ofintestinal transit by 5-HT in the distal gut also depends on anadrenergic pathway. Buffer perfusion was through both fistulas asdescribed in Example 14(12) except that 5-HT (0.05 mg/kg/h) wasadministered to the distal gut during the 90 minute perfusion period.The results are summarized in FIG. 19.

Slowing of intestinal transit by 5-HT (83.3±3.3% marker recovery minus5-HT [Buffer Control in FIG. 19] vs. 36.1±2.3% recovery withadministration of 5-HT to the distal gut [5-HT-NS in FIG. 19]) wasabolished by intravenous propranolol. In the presence of propanolol,marker recovery increased to 77.7±7.6% (5-HT-Prop in FIG. 19). Thisresult implies that the slowing of transit by 5-HT depends on apropranolol-sensitive, extrinsic adrenergic pathway, perhaps similar tothat responsible for the response to distal gut fat.

Enterochromaffin cells of the intestinal mucosa and myenteric 5-HTneurons are innervated by adrenergic nerves. (Gershon M D, Sherman D L.,Noradrenergic innervation of serotoninergic neurons in the myentericplexus, J Comp Neurol. 1987 May 8; 259(2):193-210 [1987]). To test thehypothesis that the slowing of intestinal transit by distal gut fat(ileal brake) and 5-HT depended on an adrenergic pathway, five dogs wereequipped with duodenal (10 cm from the pylorus) and midgut (160 cm fromthe pylorus) fistulas as described above. Using occluding Foleycatheters, the small intestine was compartmentalized into the proximal(between fistulas) and distal (beyond midgut fistula) one-half of gut.Buffer (pH 7.0) was perfused into the proximal gut while 60 mM oleatewas perfused into the distal gut at 2 ml/min for 90 min. Intestinaltransit across the proximal gut was compared during intravenousadministration of 50 μg/kg/h propranolol or saline. In addition, theeffect was also determined of 5-HT administered at 0.1 mg/kg/h onintestinal transit with and without i.v. propranolol. Intestinal transit(mean±SE) was measured by ^(99m)Tc-DTPA marker recovery in the output ofthe midgut fistula during the last 30 min of the 90 min experiment. Thecumulative % marker recovered was compared using ANOVA and additionalanalyses by paired t-test.

Results are shown in Table 8 below. Oleate (p<0.002) and 5-HT (p<0.005)perfused into the distal gut slowed transit through the proximal gut ascompared to buffer control. The slowing of intestinal transit by distalgut fat or 5-HT was both abolished by iv propranolol (p<0.01). Theseresults provide further evidence that the slowing of intestinal transitby distal gut fat or 5-HT depends on an adrenergic efferent nerve.

TABLE 8 Effect of 5-HT and propranolol on proximal intestinal transit.i.v. Agent Perfusate Saline (i.v.) Propranolol (i.v.) Buffer Control70.11 ± 6.51 — Oleate (Ileal brake) 26.62 ± 5.36 66.42 ± 8.26 5-HTdistal gut 28.27 ± 5.03 63.85 ± 8.76(14) Intestinal transit is slowed by norepinephrine in a 5-HT-mediatedneural pathway. Intestinal transit during buffer perfusion of theproximal and distal small intestine with intravenous norepinephrine (NE;adrenergic agent) in the presence or absence of the 5-HT receptorantagonist ondansetron was measured, to test the hypothesis that theslowing of intestinal transit also depends on an adrenergic efferentpathway. Perfusion of buffer was through both the duodenal and mid-gutfistulas (2 mL/min over 90 minutes); norepinephrine (0.12 μg/kg/h) wasadministered intravenously during the 90 minute perfusion period; andnormal saline with or without ondansetron (0.7 mg/kg/h; n=2) wasadministered in the perfusate to the proximal gut. The results aresummarized in FIG. 20.

Intestinal transit was slowed by NE so that marker recovery was reducedfrom 76.9% (Buffer Control in FIG. 20) to 13.3% (NE-NS in FIG. 20).Ondansetron abolished this slowing effect with marker recovery increasedto 63.4% (NE-Ond in FIG. 20), to implies that NE (adrenergic efferent)slows transit via a 5-HT-mediated pathway. This result confirms thatslowing of intestinal transit is mediated by an adrenergic efferentprojecting from the prevertebral ganglion to the gut action on a5-HT-mediated pathway.

To test the hypothesis that norepinephrine slows intestinal transit via5-HT3 receptors, buffer transit across the proximal gut was comparedduring intravenous administration of norepinephrine with and withoutlumenally-perfused ondansetron. Five dogs were equipped with duodenal(10 cm from the pylorus) and midgut (160 cm from the pylorus) fistulasas described above. Using occluding Foley catheters, the small intestinewas compartmentalized into the proximal (between fistulas) and distal(beyond midgut fistula) one-half of gut. Buffer (pH 7.0) was perfusedinto the proximal gut at 2 ml/min for 90 min. Intestinal transit ofbuffer across the proximal gut was compared during intravenousadministration of 50 mg norepinephrine/30 ml/1.5 h with and withoutondansetron perfused lumenally (0.7 mg/kg/h). Intestinal transit(mean±SE) was measured by ^(99m)Tc-DTPA marker recovery in the output ofthe midgut fistula during the last 30 min of the 90 min experiment. Thecumulative % marker recovered was compared using ANOVA and additionalanalyses by paired t-test.

Results are shown in Table 9 below. These results show that both anadrenergic and serotonergic pathways are involved in the slowing ofintestinal transit.

TABLE 9 Effects of norepinephrine (NE) and ondansetron (Ond) on proximalintestinal transit. Transit Across Proximal Gut (Cumulative % MarkerRecovered) Buffer Control 68.5 ± 5.0^(a) Buffer + NE 16.3 ± 3.4^(ab)Buffer + NE + Ond 63.0 ± 4.4^(b) ^(a)p < 0.003 ^(b)P < 0.0009(15) The fat-induced jejunal brake depends on the slowing effect of analoxone-sensitive, opioid neural pathway. To test the hypothesis thatthe slowing of intestinal transit depended on an opioid pathway, theproximal gut was perfused (2 mL/minute for 90 minutes) with buffercontaining 60 mM oleate and 0 (normal saline), 3, 6, or 12 mg ofnaloxone mixed therein, an opioid receptor antagonist. As shown in FIG.21, the fat-induced jejunal brake response depended on the dose ofnaloxone mixed with the oleate (p<0.05, 1-way ANOVA) (n=7).Specifically, marker recovery was 30.0±3.6% with 0 mg naloxone,41.0±5.2% with 3 mg naloxone, 62.8±8.2% with 6 mg naloxone and 60.6±6.1%with 12 mg naloxone. This result demonstrates that proximal gut fatslows intestinal transit via opioid pathway.(16) The effect of naloxone was specific for fat-triggered feedback.Intestinal transit was compared during perfusion of the proximal gutwith buffer containing 0 (normal saline) or 6 mg naloxone (n=3). Therate of intestinal transit was not significantly affected by the opioidreceptor antagonist naloxone when fat was not present in the proximalgut. Marker recovery was 88.0±1.3% with naloxone and 81.3±6.1% withoutnaloxone. This implies that the accelerating effect of naloxone wasspecific for reversing the jejunal brake effect of fat.(17) The fat-induced ileal brake depends on the slowing effect of anefferent, naloxone-sensitive, opioid neural pathway. The fistulated dogmodel allowed for the compartmentalization of the afferent limb (distalgut) from efferent limb (proximal gut) of the fat-induced ileal brake.To test for the location of the opioid pathway involved in the slowingof transit by fat, perfusion of buffer was through both the duodenal andmid-gut fistulas (2 mL/min over 90 minutes); the buffer administeredthrough the mid-gut fistula to the distal gut contained 60 mM oleate toinduce the ileal brake; 6 mg naloxone was delivered into either theproximal or distal gut (n=11). The results are summarized in FIG. 22.

Naloxone delivered to the proximal gut increased marker recovery from34.6±4.8% to 76.2±5.2% (Naloxone in Prox in FIG. 21), but naloxonedelivered to the distal gut had no effect on the ileal brake (markerrecovery of 29.4±5.4% [Naloxone in Dist in FIG. 21]). This resultimplies that the fat-induced ileal brake depends on an efferent,naloxone-sensitive opioid pathway, because an identical amount ofnaloxone was delivered into either of the two compartments, but theaccelerating effect only occurred when naloxone was delivered into theefferent compartment. Therefore, an opioid pathway is involved that islocated peripherally, rather than systemically. The accelerating effectin response to the opioid receptor antagonist is a result of theefferent location of the opioid pathway. It cannot be explained on thebasis of chemical interaction with the perfusate, since the accelerationof transit was seen when naloxone was mixed with oleate in Example14(15), as well as with buffer in this experiment.

(18) Mu and kappa opioid antagonists abolish fat-induced ileal brake.The fat-induced ileal brake (marker recovery 33.1%) was abolished by amu antagonist (H2186, Sigma) delivered into the proximal gut so thatmarker recovery increased to 43.8% at 0.037 mg H2186, 88.2% at 0.05 mgH2186 and 66.8% at 0.1 mg H2186 over 90 minutes. A similar effect wasseen when a kappa antagonist (H3116, Sigma) was used (marker recoveryincreased to 73.2% at 0.075 mg H3116, 90.9% at 0.1 mg H3116, and 61.8%at 0.125 mg H3116 over 90 minutes; n=1).(19) Slowing of intestinal transit by distal gut 5-HT depends on analoxone-sensitive, opioid neural pathway. In Example 14(5), 5-HT in thedistal gut slowed intestinal transit, similar to the effect of fat inthe distal gut. Since the ileal brake induced by fat in the distal gutwas shown to depend on an efferent, naloxone-sensitive opioid pathway(Example 14(17), it was tested whether the slowing of intestinal transitin response to 5-HT in the distal gut also depends on an efferent,opioid pathway. Buffer was perfused into both the proximal and distalguts at 2 mL/minute for 90 minutes. Either normal saline (Buffer Controlin FIG. 23) or 5-HT (0.05 mg/kg/h; 5-HT in Dist in FIG. 23) wasadministered to the distal gut over the 90 minute perfusion. When theperfusate to the distal gut contained 5-HT (i.e., 5-HT in Dist),naloxone (6 mg) was simultaneously delivered through the duodenalfistula to the proximal gut over the 90 minutes (Naloxone in Prox inFIG. 23). Results are summarized in FIG. 23.

First, intestinal transit was slowed by 5HT in the distal gut. Markerrecovery was reduced from 79.4±4.1% (Buffer Control) to 37.0±1.8% (5-HTin Dist). Second, naloxone in proximal gut abolished this slowing effectwith marker recovery increased to 90.1±4.6% (Naloxone in Prox). Theseresults imply that slowed intestinal transit in response to 5-HT in thedistal gut, depends on an efferent opioid pathway.

The foregoing examples being illustrative but not an exhaustivedescription of the embodiments of the present invention, the followingclaims are presented.

1. A method of treating small intestinal bacterial overgrowth (SIBO) ora SIBO-caused condition in a human subject, said method comprising:detecting in the subject by suitable detection means, the presence ofSIBO, wherein a population of proliferating bacteria is present in thesmall intestine of the subject, or detecting with said means the absenceof SIBO; and, if the presence of SIBO is detected in the subject,depriving the bacterial population of nutrient(s) sufficiently toinhibit the growth of said bacteria in the small intestine, and therebyat least partially eradicating SIBO in the human subject, wherein theSIBO-caused condition is selected from the group consisting of irritablebowel syndrome, fibromyalgia, chronic pelvic pain syndrome, chronicfatigue syndrome, depression, impaired mentation, impaired memory,halitosis, tinnitus, sugar craving, autism, attentiondeficit/hyperactivity disorder, drug sensitivity, an autoimmune disease,and Crohn's disease.
 2. The method of claim 1, wherein the autoimmunedisease is systemic lupus erythematosus or multiple sclerosis.
 3. Themethod of claim 1, further comprising: in the presence of SIBO in thehuman subject, causing the subject to consume, for a sustained period, adiet consisting essentially of nutrients that upon arrival in the uppergastrointestinal tract of the subject, are at least partiallypredigested, said sustained period being sufficient to at leastpartially eradicate SIBO in the human subject.
 4. The method of claim 3,wherein the period is at least about three days.
 5. The method of claim3, wherein the at least partially predigested nutrients are contained ina comestible total enteral nutrition formulation.
 6. The method of claim3, further comprising: administering to the subject a pancreatic enzymesupplement 0 to 60 minutes before a meal, such that nutrients containedin said meal are at least partially predigested in the uppergastrointestinal tract of the subject by the activity of said pancreaticenzyme supplement.
 7. The method of claim 1, further comprising: in thepresence of SIBO in the human subject, enhancing the digestion and/orabsorption of the nutrient(s) in the upper gastrointestinal tract ofsaid human subject by slowing transit of said nutrients across the uppergastrointestinal tract of said human subject, thereby at least partiallydepriving the bacterial population of the nutrient(s).
 8. The method ofclaim 7, further comprising: administering a pharmaceutically acceptablecomposition to said human subject by an oral or enteral delivery route,said human subject having an intrinsic cholinergic afferent neuralpathway projecting from a peptide YY-sensitive primary sensory neuron inthe intestinal wall of said subject to a prevertebral celiac ganglionand having an adrenergic efferent neural pathway projecting from saidganglion to one or more enterochromaffin cells in the intestinal mucosaand/or to a serotonergic interneuron linked in a myenteric plexus and/orsubmucous plexus to an opioid interneuron, said opioid interneuron alsobeing linked by an intestino-fugal opioid pathway projecting to saidganglion, with one or more neural connections to the central nervoussystem and back to the gut projecting from the ganglion, saidpharmaceutically acceptable composition comprising an active agent, saidactive agent being selected from the group consisting of (A) activelipids; (B) serotonin, serotonin agonists, or serotonin re-uptakeinhibitors; (C) peptide YY or peptide YY functional analogs; (D)calcitonin gene-related peptide or functional analogs thereof; (E)adrenergic agonist; (F) opioid agonists; (G) combinations of any of (A),(B), (C), (D), (E) and/or (F); and (H) antagonists of receptors for anyof (B), (C), (D), (E) and/or (F), said active agent being delivered inan amount and under conditions such that the cholinergic intestino-fugalpathway, at least one prevertebral ganglionic pathway, the adrenergicefferent neural pathway, the serotonergic interneuron and/or the opioidinterneuron are activated by the action of any of (A) through (G),whereby the rate of upper gastrointestinal transit in the subject isslowed, thereby enhancing the digestion and/or absorption of thenutrient(s) in the upper gastrointestinal tract of said human subject.9. The method of claim 7, further comprising: administering agastrointestinal transit-slowing composition comprising a carrier and adispersion consisting essentially of an active lipid in the carrier, theactive lipid being selected from the group consisting of saturated andunsaturated fatty acids, fully hydrolyzed fats and mixtures thereof, inan amount and in a form effective to promote contact of the lipid withthe subject's small intestine and thereby slow gastrointestinal transitand at least partially eradicate SIBO in the human subject.
 10. Themethod of claim 9, wherein the active lipid is selected from the groupconsisting of: (A) caprolic acid, caprulic acid, capric acid, lauricacid, myristic acid, oleic acid, palmitic acid, stearic acid,palmitoleic acid, linoleic acid, linolenic acid, trans-hexadecanoicacid, elaidic acid, columbinic acid, arachidic acid, behenic acideicosenoic acid, erucic acid, bressidic acid, cetoleic acid, nervonicacid, Mead acid, arachidonic acid, timnodonic acid, clupanodonic acid,or docosahexaenoic acid; (B) pharmaceutically acceptable salts of any of(A); and (C) mixtures of any of (A) or (B).
 11. The method of claim 10,wherein the active lipid comprises oleic acid or a pharmaceuticallyacceptable oleate salt.
 12. The method of claim 9, wherein the activelipid comprises fully hydrolyzed fats.
 13. The method of claim 9,wherein the active lipid comprises a fatty acid or a pharmaceuticallyacceptable salt thereof.
 14. The method of claim 9, wherein the activelipid is: (A) a fatty acid selected from the group of (C4-C24) saturatedand unsaturated fatty acids; (B) a pharmaceutically acceptable salt ofany of (A); or (C) a mixture of any of (A) and/or (B).
 15. The method ofclaim 9, wherein the fatty acid comprises oleic acid, a pharmaceuticallyacceptable oleate salt, or a mixture of either of these with other fattyacids or salts thereof.
 16. The method of claim 8, wherein oraladministration is by ingestion of coated or uncoated microspheres orparticles, of a dispersible powder or granule formulation, of asuspension, emulsion, solution, syrup, or elixir, or of a coated oruncoated tablet, troche, capsule, caplet, or lozenge.
 17. The method ofclaim 9, wherein oral administration is by ingestion of coated oruncoated microspheres or particles, of a dispersible powder or granuleformulation, of a suspension, emulsion, solution, syrup, or elixir, orof a coated or uncoated tablet, troche, capsule, caplet, or lozenge. 18.The method of claim 8, wherein the active agent is selected from thegroup consisting of serotonin, serotonin agonists, serotonin re-uptakeinhibitors, 5-HT3 receptor antagonists, and 5-HT4 receptor antagonists.19. The method of claim 18, wherein the active agent is serotonin, andthe serotonin is administered to the human subject, 0 to 60 minutesbefore a meal, in an amount from about 0.03 to about 0.1 mg/kg bodymass.
 20. A method of treating small intestinal bacterial overgrowth(SIBO) or a SIBO-caused condition in a human subject, said methodcomprising: detecting in the subject by suitable detection means, thepresence of SIBO, wherein a population of proliferating bacteria ispresent in the small intestine of the subject, or detecting with saidmeans the absence of SIBO; and, if the presence of SIBO is detected inthe subject, introducing into the lumen of the small intestine of thesubject a pharmaceutically acceptable disinfectant composition in anamount sufficient to inhibit the growth of said bacteria in the smallintestine, and thereby at least partially eradicating SIBO in the humansubject, wherein the SIBO-caused condition is selected from the groupconsisting of fibromyalgia, chronic pelvic pain syndrome, chronicfatigue syndrome, depression, impaired mentation, impaired memory,halitosis, tinnitus, sugar craving, autism, attentiondeficit/hyperactivity disorder, drug sensitivity, an autoimmune disease,and Crohn's disease.
 21. The method of claim 20, wherein thepharmaceutically acceptable disinfectant composition consistsessentially of (A) hydrogen peroxide, (B) a bismuth-containing compound,(C) an iodine-containing compound, or (D) a salt of (B) or (C).
 22. Themethod of claim 20, wherein the autoimmune disease is systemic lupuserythematosus or multiple sclerosis.
 23. A method of treating smallintestinal bacterial overgrowth (SIBO) or a SIBO-caused condition in ahuman subject, said method comprising: detecting in the subject bysuitable detection means, the presence of SIBO, wherein a population ofproliferating bacteria is present in the small intestine of the subject,or detecting with said means the absence of SIBO; and, if the presenceof SIBO is detected in the subject, administering to the subject apharmaceutically acceptable composition comprising a stabilizer of mastcell membranes in the lumenal wall of the small intestine, in an amountsufficient to inhibit a mast cell-mediated immune response in the humansubject, wherein the SIBO-caused condition is selected from the groupconsisting of fibromyalgia, chronic pelvic pain syndrome, chronicfatigue syndrome, depression, impaired mentation, impaired memory,halitosis, tinnitus, sugar craving, autism, attentiondeficit/hyperactivity disorder, drug sensitivity, an autoimmune disease,and Crohn's disease.
 24. The method of claim 23, wherein the autoimmunedisease is systemic lupus erythematosus or multiple sclerosis.
 25. Themethod of claim 23, wherein the stabilizer of lumenal mast cells isoxatamide or chromoglycate.